慢性牙周炎对OLETF大鼠肾脏病变的影响及其机制的探讨
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摘要
研究背景
近年来,随着国内牙周医学的快速发展,越来越多的口腔医生并始关注牙周疾病,尤其是发病率极高的慢性牙周炎。美国人慢性牙周炎的发病率约47.6%,中国人群其发病率更高。众所周知,慢性牙周炎是菌斑微生物引起的慢性非特异性炎症性疾病,以造成牙周组织破坏、牙槽骨吸收和病理性牙周袋的行为为特征。牙周炎是一种炎症性疾病,但是我们不能再将其看作是一种局限于局部炎症,它可能通过增加机体全身炎症负担和增强机体免疫反应的途径来影响全身其它系统。
目前,有大量研究证明了牙周炎和全身各系统的疾病相关联。其中它与2型糖尿病(type2diabetes mellitus T2DM)的研究引起极大关注。T2DM是一种危害极大、发病率极高的慢性病。随着老龄化的进程,糖尿病合并牙周炎的患者急剧增加。现已证明,牙周炎与T2DM互为对方的独立危险因素。慢性牙周炎可加重机体全身炎症状态,升高全身炎症因子水平,促进胰岛素抵抗,不利于血糖控制,进而影响T2DM及其并发症的发生和发展。而T2DM时,机体对炎症的免疫力增强,牙周组织局部分泌过多的炎性细胞、炎性因子和蛋白酶,使得局部组织的破坏增加,加重牙周炎症程度。
而2型糖尿病其最大的危害在于其远期的慢性并发症,其中之一糖尿病肾病(Diabetic Kidney Disease DKD)是糖尿病患者的主要死亡原因之一。DKD的主要病理改变为肾脏细胞外基质-Ⅳ型胶原(胶原)的过度蓄积,产生肾小球硬化和肾间质纤维化。导致产生以上病变的机制复杂,但涉及几个关键的细胞因子和蛋白酶：转化生长因子β1(transforming growth factor--β1TGF-β1)在肾间质纤维化和肾小球硬化中起着关键作用；基质金属蛋白酶系统中MMP-2(明胶酶A),MMP-9(明胶酶B)以及抑制因子TIMP-1是研究发现主要调节肾脏细胞外基质(extracellular matrix ECM)平衡的关键因子。
目前对于牙周炎对T2DM的慢性并发症DKD影响的研究非常少,且多为临床病例的观察。因此本研究拟采用应用价值极高,且与人类T2DM发病极其相似的自发型2型糖尿病动物模型OLETF大鼠,在其上复合慢性牙周炎建立两种疾病复合动物模型的方法,排除其他混杂因素的干扰,初步探讨牙周炎对T2DM发生发展的作用。分析T2DM时牙周炎是否会对肾脏功能和肾脏组织结构产生影响。并通过检测与DKD发生密切相关的Ⅳ型胶原、TGF-β1、MMP-2, MMP-9, TIMP-1蛋白表达和基因转录水平的差异,进一步分析牙周炎对T2DM大鼠肾脏病损是否存在影响及其影响的病理机制,为进一步研究牙周炎与T2DM及DKD的相互关系建立可靠的实验基础,为临床多学科联合诊断,治疗和预防提供证据。
第一章OLETF大鼠慢性牙周炎模型的建立
目的
在OLETF大鼠到达糖耐量受损期时,通过牙周局部丝线结扎联合牙周局部涂菌建立糖尿病复合牙周炎模型,为下一步研究牙周炎对T2DM大鼠肾脏功能及肾脏病损是否存在影响及其影响的病理机制奠定基础。
方法
(1)自发性2型糖尿病OLETF大鼠(O组),4周龄,雄性,30只；同种系同周龄糖耐量正常的LETO大鼠(L组),雄性,20只。每4周行一次口服糖耐量检测,按2g/kg予50%葡萄糖溶液灌胃,依据OGTT试验的结果判定,血糖峰值>16.7mmol/L和葡萄糖灌胃后120min血糖>11.1mmol/L诊断为糖尿病,具备上述条件之一者为糖耐量受损(Impaired Glucose Tolerance IGT))。36周龄时,将到达糖耐量受损期(IGT期)OLETF大鼠26只,随即分为两组,一组附加牙周炎(O CP(+)组)(n=13),另外一组对照(O CP(-)组)(n=13)；LETO大鼠也随机分为两组,一组附加牙周炎(L CP(+)组)(n=10),另一组为正常对照(L CP(-)组)(n=10)。
(2)CP(+)组大鼠给予丝线分别结扎于左、右上颌第一磨牙(molar1, M1)及第二磨牙(molar2, M2)牙颈部,并在结扎处牙周涂抹四种牙周常见致病菌混悬液。定期检查丝线,有脱落者及时重新结扎并涂菌。
(3)每4周行一次OGTT,测体重,观察其体重和血糖的变化。并在36周龄和46周龄时,尾静脉采血测定其血清胰岛素水平的动态变化并计算糖尿病人胰岛素抵抗稳态模型(Homeostasis model assessment-insulin resistance HOMA-IR)值。
(4)56周龄时,处死所有实验大鼠,采集血清检测大鼠血脂水平；收集组织样本,颌骨样本采用Micro-CT扫描,EDTA脱钙后HE染色,观察牙周炎症状态。
四组之间的整体比较采用2×2析因设计资料的方差分析,两组间单因素数据比较采用独立样本t检验；同一处理因素两个不同水平之间以及同一水平不同处理因素之间的比较采用两独立样本的t检验。不同时间段多次测量的指标,总体各组和组内都用用重复测量的方差分析。同一时间同一组内有无干预的比较用两独立样本的t检验。
结果
本实验采用与人类T2DM发病及其相似的自发性T2DM大鼠OLETF大鼠,和其正常对照LETO大鼠作为研究对象。在36周龄时到达IGT期,模拟了人类T2DM的慢性发病的过程。
OLETF大鼠表现为多食多饮多尿,倦怠少动,体重快速增加。随着实验时间延长,体重差异进一步加大。O CP(+)组、O CP(-)组随着大鼠周龄的增加,血糖AUC显著增加有统计学意义(F=5.952P=0.029),但在O组任意周龄,O CP(+)组比O CP(-)组AUC略有增高,但并无显著差异。O组大鼠血清甘油三酯显著高于L组(t=11.324P=0.002),O CP(+)组比O CP(-)组甘油三酯水平有增高,并无显著差异(t=1.031P=0.320)。O组大鼠血清胆固醇显著高于L组(t=39.089P=0.000),O CP(+)组比O CP(-)组甘油三酯水平显著增高(t=2.532P=0.024),并且糖尿病和牙周炎二者之间存在交互作用(F=4.743P=0.038)
O组血清FI浓度和HOMA-IR值均显著高于L组。但O CP(-)组FI水平略高于O CP(+)组。O CP(-)组和O CP(+)组随着周龄增加HOMA-IR值的增加有显著性(F=38.233P=0.000<0.01),且同周龄时O CP(+)组比O CP(-)组HOMA-IR值高,但并无统计学差异。
牙周结扎大鼠口内检查发现牙颈部结扎处有食物残留,牙龈红肿、牙槽骨吸收、牙周袋形成,在O CP(+)组牙周炎的炎症状态更重；Micro-CT的三维重建显示四组大鼠牙槽骨吸收体积,O CP(+)组与L CP(+)组牙槽骨骨丧失量明显分别高于O CP(-)组和L CP(-)组,差别有统计学意义(P=0.013,P=0.004),且O CP(+)组骨丧失量要多于L CP(+)组骨丧失量。O组牙槽骨吸收体积总体上明显多于L组(t==11.476P=0.010)。
脱钙大鼠颌骨组织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。在OCP(+)组,炎症更重,可见结合上皮大幅根向迁移,牙周袋内及牙龈结缔组织内大量弥漫炎细胞浸润,甚至大量炎细胞游走到牙周袋内。
结论
OLETF大鼠在糖脂代谢和胰岛素抵抗方面可以较好的模拟人类T2DM的发病特征,是非常典型的T2DM动物模型。在OLETF大鼠附加牙周炎之后,牙周炎可促进胰岛素抵抗的程度和进度、不利于血糖控制,血清胰岛素代偿性增高,血脂增高,进而恶化T2DM的进程。T2DM也可加重牙周组织破坏,牙周炎症程度明显比单纯牙周炎组重。
第二章慢性牙周炎对大鼠肾功和肾脏组织形态的影响
目的
通过在OLETF大鼠附加牙周炎,探讨慢性牙周炎是否对OLETF肾脏功能和肾脏组织形态学存在影响。为探讨牙周炎是否影响T2DM并发症DKD的发生发展提供病理基础。
方法
收集大鼠处死时血清,全自动生化分析仪测大鼠血清反映肾功的生化指标,包括血清白蛋白(Albumin ALB)、总蛋白(Total protein TP)、尿素(Urea)肌酐(Creatinine Cr)四个指标。取单侧肾脏,肾门处纵剖肾脏组织常规制作组织2-3μm切片,分别行常规HE染色和PAS、MASSON、PASM特殊染色,结合起来全面分析肾脏病理改变。通过分析PAS染色切片计算肾小球硬化指数(glomerulosclerosis index GSI),利用MASSON染色计算肾间质纤维化指数(renal interstitial fibrosis index RIFI),比较各组间大鼠以上指标的差异。实验结果用析因设计的方差分析和两独立样本的t检验。
结果
O组比L组血清总蛋白和白蛋白水显著下降(t=11.131P=0.002)。一致的是O CP(+)组比O CP(-)组血清ALB和TP的水平均有显著下降。而在L组有无CP干预,二者之间的比较没有明显差异。O组大鼠血清肌酐水平均显著高于L组(t=9.099P=0.005)。L组大鼠有无CP干预肌酐水平无明显变化。O CP(+)组大鼠血清肌酐水平明显高于O组,差异有统计学意义(t=4.443P=0.000<0.01)。血清尿素结果显示O组比L组显著升高(P=0.024)。L CP(-)与L CP(+)组尿素水平接近。而O CP(+)组比O CP(-)尿素升高明显,但并无统计学差异。(t=1.484,P=-0.160)。
L CP㈩组组织学特征与L组接近,表现为正常肾脏的组织学特点。O组大鼠肾小球直径增大,鲍曼氏囊囊壁增厚明显,肾小球球囊部分粘连,可见囊腔扩大。肾小球系膜基质表现为中度至重度增生,呈弥漫性或结节状,再加上肾小球毛细血管基底膜增厚,部分区域出现局灶性肾小球硬化；肾小球周围出、入球小动脉管壁增厚。部分肾小管扩张,管腔内可见蛋白样物质或蛋白管型。肾小管上皮细胞玻璃样或空泡变性,部分区域甚至可见肾小管上皮萎缩、坏死、脱落,导致肾小管正常结构消失,晚期被纤维组织替代。间质可见散在炎细胞,严重者可见淋巴细胞和单核细胞成灶性浸润,肾间质纤维化。两种疾病的复合组病变程度更重。
O组大鼠肾GSI和RIFI均显著高于L组大鼠(t=85.112P=0.000<0.01,t=27887.6P=0.000<0.01)。在L CP(+)组,GSI略高于L CP(-)组,二者之间没有显著性差异(P=0.076)。OCP(+)组大鼠GSI与OCP(-)组比较有显著性差异(P值均<0.01)。而且T2DM和牙周炎对大鼠GSI存在交互效应(F=75.060,P=0.000<0.01)。RIFI也是相似的结果,在L CP(+)组,RIFI也略高于L CP(-)组,但二者之间没有显著性差异(P=0.296)。O CP(+)组大鼠与OCP(-)组比较有显著性差异(t=23.709P<0.01)。而且T2DM和牙周炎对大鼠RIFI存在交互效应(F=477.875,P=0.000)。
值得注意的是炎症浸润特征在O组很明显。在O CP(-)组8只大鼠组织切片中发现3只大鼠有大量散在炎细胞浸润或者大灶性炎细胞浸润(3/8),而在OCP㈩组中,有大量散在炎细胞浸润或者大灶性炎细胞浸润的大鼠例数明显增加,为6只(6/8),且炎细胞大多是单核细胞和巨噬细胞。
结论
本研究发现慢性牙周炎未对正常大鼠肾脏结构和功能产生显著影响；但在T2DM肾脏已有损害的情况下,慢性牙周炎显著增加了肾间质炎细胞浸润的风险,其促进了肾小球硬化、肾小管破坏及肾间质纤维化,加重了肾脏损害,恶化了肾功能,并且T2DM和牙周炎在肾脏病变发生中有交互、协同作用。
第三章慢性牙周炎对OLETF大鼠肾脏病变调控蛋白及其mRNA表达的影响
目的
检测各组大鼠肾脏组织中Ⅳ型胶原、TGF-β1、MMP-2, MMP-9, TIMP-1蛋白和基因转录水平的表达差异。分析牙周炎是否对肾脏组织代谢密切相关的关键物质的蛋白表达和基因转录水平产生了影响,进而探讨T2DM时慢性牙周炎影响肾脏损害可能的病理机制。
方法
采用免疫组化ENVISION二步法检测四组大鼠肾脏组织中Ⅳ型胶原、TGF-β1、MMP-2, MMP-9, TIMP-1蛋白的表达,进行半定量分析；ELISA法检测大鼠血清中TGF-β1的浓度；另采用实时定量PCR(Real-Time PCR)技术的Sybr green (荧光染料掺入)法来进行各组大鼠肾脏组织中Ⅳ型胶原、TGF-β1、MMP-2、MMP-9、TIMP-1、的mRNA基因表达差异分析。实验结果用析因设计的方差分析和两独立样本的t检验。
结果
IV型胶原蛋白表达于肾小球基底膜和系膜基质,血管壁、肾小管基底膜等部位。O组表达明显高于L组,且O CP(+)组与O CP(-)组之间表达量有统计学差异。(t=-11.099P=0.000<0.01),而L CP(+)组与L CP(-)组之间表达没有差异(t=2.032P=0.062)。
TGF-β1蛋白表达较广泛,主要集中在近曲小管上皮胞浆的部位,另外远曲小管上皮胞浆也有中等阳性的表达；肾小球基质和内皮的部位可见极少量表达。四组大鼠中TGF-β1均有表达,L组表达较低,O组比L组表达显著增强(t=2034.01P=0.000<0.01)。两组内在CP干预后其表达都明显增强。分组和牙周炎对TGF-β1蛋白的表达都有显著影响,并且牙周炎和糖尿病对TGF-β1蛋白的表达有交互相应(F=5.729P=0.024)。TGF-β1mRNA表达趋势与蛋白表达趋势一致。L组附加CP后其表达显著增高(t=4.851P=0.000<0.01)。
MMP-9蛋白主要表达于近曲小管和远曲小管上皮细胞,少量表达于系膜细胞。正常对照L组MMP-9的表达要明显高于O组(t=297.868P=0.000<0.01),L CP(+)组与L CP(-)组相比表达明显降低(t=11.274P=0.000<0.01)。O CP(-)组MMP-9蛋白表达的值与O CP㈩组接近,没有统计学差异(t=1.885,P=0.085)。T2DM和牙周炎之间有交互效应(F=66.397P--0.000)。四组大鼠肾脏MMP-9mRNA表达与其蛋白的表达总体趋势也一致,在正常L组大鼠肾脏都有高水平表达,而O组显著低于L组。但基因表达上O CP(+)组明显低于O CP(-)组(t=4.360P=0.001)。
MMP-2蛋白主要表达于近曲小管上皮细胞胞浆,远曲小管和肾小球可见微量表达,还可以发现在炎症浸润区域,炎细胞有少量表达。表达量最高的仍然是L两组,L CP㈩组与L CP(-)组相比表达明显降低(t=9.030P=0.000<0.01)。O组比L组显著降低(t=3184.226P=0.000<0.01)。O CP(+)组MMP-2蛋白表达值最低,阳性着色区域不明显。主效应分组和牙周炎对MMP-2蛋白的表达都有显著影响,主效应分组和牙周炎之间有交互效应(F=13.529P=0.001)。四组大鼠肾脏MMP-2mRNA表达与其蛋白的表达趋势正好相反。MMP-2基因表达O组显著高于L组(P<0.01),并且O CP(+)组的表达显著高于O CP(-)组(t=2.475P=0.027)。
TIMP-1蛋白表达于近曲小管、远曲小管、肾间质和肾小球基质区域。O组TIMP-1表达明显高于L组(t=1721.404P=0.000<0.01),O CP(+)组比O CP(-)组表达明显增强(t=9.007P=0.000<0.01),L CP㈩组与L CP(-)组相比表达接近,没有统计学差异(t=1.245P=0.234)。
四组大鼠肾脏TGF-β1, TIMP-1和IV型胶原mRNA表达与各自蛋白的表达趋势一致,即在L组大鼠肾脏都有正常的低水平表达,而以上三者在O组表达量比L组表达都显著上调(P<0.01),并且在O CP㈩组表达都略高于O CP(-)组,差异均没有统计学意义。
结论：
牙周炎复合糖尿病时,促进了肾脏组织以及血清中促纤维化的核心因子TGF-β1的表达增高,也促进了抑制胶原降解的酶TIMP-1的高表达；却抑制了细胞外机制降解酶MMP-2,MMP-9的正常表达,从而导致了肾脏组织IV型胶原沉积增多。
糖尿病状态下,肾脏细胞外基质的代谢平衡已经被打破,胶原生成增多,降解减少；而在糖尿病复合牙周炎后,这种不平衡更加恶化,牙周炎加剧了细胞外基质的代谢失衡,加速肾小球硬化和肾间质纤维化,促进肾脏功能的进一步丧失。
Background
In recent years, with the rapid progress of periodontal medicine, more dentists are beginning to pay much more attention to periodontal disease, especially the chronic periodontitis. Periodontitis is a common infection of the periodontal tissues and a major cause of tooth loss in adults. The most prevalent form is chronic periodontitis. The chronic periodontitis incidence rate in Americans is about47.6%in2010. Even the worse is the higher incidence in the Chinese population. As we all know, chronic periodontitis is a kind of non-specific inflammatory diseases caused by plaque microorganisms. That would result in periodontal tissue destruction, which is characterized by alveolar bone resorption and pathological periodontal pocket. Periodontitis is an inflammatory disease, but we would no longer regard it as a limited local inflammation. It might influence the whole body by the ways to increase the body's systemic inflammatory burden and enhance the immune response.
Currently, there are a large number of studies have revealed periodontitis and systemic disease associated, with type2diabetes (T2DM) is of great concern. T2DM is a kind of chronic diseases with high incidence and great harm. With the process of aging, diabetes and periodontitis patients have increased dramatically. It has been revealed that periodontitis and T2DM is the independent risk factor for each other. Chronic periodontitis may increase the body's systemic inflammatory burden, increased systemic levels of inflammatory factors, and promote insulin resistance. And that was not conducive to blood glucose control, thereby affecting the occurrence and development of T2DM and its complications. In T2DM, the enhancement of the immune system to the body inflammation leads to local excessive infiltration of inflammatory cells in periodontal tissues, excessive secretion of inflammatory factors and proteases. That would cause the more destruction in local tissue and increase the degree of periodontal inflammation.
The greatest hazards of T2DM are the long-term chronic complications. Diabetic kidney disease (DKD) is one of them and it is the leading cause of death of patients with T2DM. The main pathological changes of DKD are the renal extracellular matrix-type IV collagen (collagen) excessive accumulation, resulting in glomerulosclerosis and renal interstitial fibrosis. Mechanisms leading to the kidney lesions are very complex. Involving several key cytokines and proteases:TGF-β1plays a key role in renal interstitial fibrosis and glomerulosclerosis; matrix metalloproteinase MMP-2(gelatinase A), MMP-9(gelatinase B), and inhibitor TIMP-1were found to be the key factors to regulate renal ECM balance.
Until now, there are few researches on the effect of Periodontitis on the DKD. Only a few available studies were the observation of clinical cases. Therefore, this study intends to composite type2diabetes and chronic periodontitis disease in an animal model. We aimed to discusse the the role of periodontitis in the development of T2DM and to determine whether periodontitis impact renal function and renal structure by excluding the interference of any other confounding factors. And through testing the above mentioned DKD closely related type IV collagen, TGF-betal, MMP-2, MMP-9, TIMP-1protein and Mrna expression, we attempted to further analyze whether periodontitis had an impact on DKD and the possible pathological mechanisms. And try to establish a reliable experimental basis for further study of periodontitis and T2DM or DKD relationship, diagnosis, treatment and prevention for clinical Multidisciplinary combination therapy.
CHAPTER ONE The animal model of chronic periodontitis in OLETF rats
Objective:To establish experimental periodontitis in OLETF rats by ligature and periodontal pathogen infection for further evaluating the effects of periodontal inlammation on the diabetic kidney disease and the pathogenesis in OLETF rats.
Methods:
(1)30OLETF rats (O group),4-week-old, male, spontaneously type2diabetic;20LETO rats (L group), male, with the same germline and the same age but having normal glucose tolerance, as the control group. Every4weeks, oral glucose tolerance was determined with50%glucose solution2g/kg orally administered. Diabetes diagnosis is based on the results of the OGTT test, blood glucose peak>16.7mmol/L and glucose and blood glucose>11.1mmol/L in120min as the criteria. Meeting either one of the above criteria is recognized as impaired glucose tolerance (IGT). At36weeks old,(being IGT state)26OLETF rats were randomly divided into two groups:periodontitis group (O CP (+))(n=13) and non-periodontitis group (O CP (-) group)(n=13); LETO rats were also randomly divided into two groups, periodontitis group (L CP (+) group)(n=10) and non-periodontitis group (L CP (-) group)(n=10).
(2) The bilateral maxillary first and second molars in the [L CP (+)] group and [O CP (+)] group were ligatured with3/0silks soaked with periodontal pathogens, in subgingival position for28weeks. The ligatures were checked regularly and re-done if necessary.
(3) OGTT was conducted every four weeks and measured body weight at the same time. The rat tail vein blood samples were obtained at36weeks and46weeks of age to evaluate the dynamic changes of serum insulin levels and HOMA-IR score.
(4) At56weeks 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 periodontal tissue 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℃.
The overall comparisons between the four groups were done by using a2x2factorial design analyzes. Single factor data of the two groups were compared using independent samples t test. Independent samples t test were also used to compare the same factors at two different levels and the same level of different factors. Multiple indicators measured in different times between general groups or within groups were compared by by analysis of variance with repeated measurements. With or without the intervention of comparison in the same time within the same group was compared by the two independent samples t test.
Results
The spontaneous T2DM rats-OLETF rats and normal control LETO rats were used as research objects in this study. OLETF reached the IGT period at36weeks age vividly simulating human T2DM chronic disease process.
OLETF rats showed typical clinical features of the T2DM, just like polyphagia, polydipsia and polyuria, rapid weight gain. Blood glucose AUC of the OLETF rats increase statistically significant with ageing (F=5.952P=0.029). However, at any weeks of age, AUC slightly increased in O CP (+) group than in the O CP (-) group without significant difference. Serum triglyeride (TG) and cholesterol (CHOL) in O group were significantly higher than those in Group L. TG and CHOL in O CP (+) group were both higher than those in O CP (-) group, while serum CHOL difference with significance.
Serum FI and HOMA-IR in O group was significantly higher than that in Group L. O CP (-) group FI level was slightly higher than O CP (+) group. O CP (-) group and O CP (+) group had significantly increased HOMA-IR values with increasing age.
Intraoral Periodontal examination in CP (+) group show:food residue around tooth neck, swollen gums, alveolar bone resorption, periodontal pocket. O CP (+) group had heavier periodontal inflammation. Micro-CT three-dimensional reconstruction of the rat alveolar bone in four groups display that the alveolar bone loss in O CP (+) group and L CP (+) group were significantly higher than that in O CP (-) group and L CP (-) group, respectively. Moreover, the rats in O CP (+) group have more bone loss than L CP (+) group.
Through HE staining of decalcified rat mandible, we found periodontal attachment loss, destruction of collagen fibers, inflammatory cell infiltration in the end the periodontal pocket, alveolar bone resorption significantly. In the OCP (+) group, inflammation was more heavier, with junctional epithelium migrate to root direction, a large number of diffuse inflammatory cell infiltration in periodontal pockets and gingival connective tissue, and even migrate into the periodontal pocket.
Conculusion
OLETF rat is a very typical T2DM animal model which simulates human T2DM characteristics in glucose and lipid metabolism and insulin resistance vividly.
Periodontitis promote the degree and progress of insulin resistance in T2DM. It could deteriorate glycemic control and lead to compensatory high serum insulin. Thereby deteriorate the T2DM state. T2DM could aggravate periodontal tissue destruction conversly. The degree of periodontal inflammation in T2DM is much heavier than in normal periodontitis group.
Chaper2Effect of Chronic periodontitis on rat renal function and renal morphology
Objective
We aimed to investigate whether chronic periodontitis affect the OLETF kidney function and the morphology kidneys by measuring kidney function indices and observing the kidney morphology. To supply evidence for the Pathological basis of whether periodontitis affecting the development of T2DM complications.
Methods
The biochemical markers in the four rat groups including serum albumin (ALB), total protein (TP), urea (UREA) and creatinine (Cr) were analyzed by automatic biochemical analysis instruments. In order to do a comprehensive analysis of renal pathological changes, routine HE staining and PAS, MASSON, PASM special staining were adiministered. Glomerulosclerosis index (GSI) was calculated by analyzing the PAS stained sections. MASSON staining was used to calculate the index renal interstitial fibrosis (RIFI). Factorial design analysis of variance and two independent sample t-test were used for statistical analysis of the results.
Results
The levels of serum TP and ALB decreased significantly in O group than in the group L (t=11.131, P=0.002). Consistently, serum levels of ALB and TP decreased significantly in O CP (+) group than in O CP (-). In the L group or without CP intervention, there is no significant difference. Serum creatinine and urea levels were significantly higher in O gourps than in L groups (t=9.099, P=0.005).
The histological features in L CP (+) group were close to L group showing normal renal histological features. The glomerular diameter increased and cortex narrowed and medulla widened in OLETF rats. Bowman's capsule wall became thickened significantly, glomerular balloons were adhered partly and cysts increased. There are moderate to severe hyperplasia in glomerular mesangial matrix diffusively or nodularly, basement membrane thickening of glomerular capillary, focal glomerulosclerosis in some areas, glomerular capillary plexus lobulation, and vascular wall thickening of arteryies in and out of glomerular surrounding. There were hyaloid and vacuolar degeneration in cortical tubular epithelial cells, dilatation of some tubular, protein-like substances or protein casts in lumen. It was also found that there were tubular epithelial cell desquamation, tubular degeneration and atrophy in some areas, dilation or atrophy of hylic tubules, scattered or gathering infiltration of lymphocytes and mononuclear cells and fibrosis in interstitial areas.
Concerning the GSI and RIFI, their results are similar. The GSI and RIFI in OLETF rats are significantly higher than Leto rats (P<0.01). They are slightly higher in L CP (+) group than in L CP (-) group, no significant difference. However, they are significantly higher in OCP (+) rat than in (P<0.01). There is an interaction effect between T2DM and periodontitis.
It is noteworthy that obvious inflammatory infiltration was found in the O group. Among O CP (-) group, a large number of scattered inflammatory cell infiltration or large focal inflammatory cell infiltration were found in three of eight rats (3/8). In O CP (+) group, a large number of scattered inflammation cell infiltration or large focal inflammatory cell infiltration in rat patients was found in six of eight rats (6/8) and most of the inflammatory cells are monocytes and macrophages.
Conclusion
Chronic periodontitis may not cause significant changes in kidney function and histopathology in normal rats. However, chronic periodontitis may significantly increase the renal interstitial inflammatory cell infiltration risk in T2DM. Moreover, the chronic periodontitis promote kidney glomerulosclerosis and renal interstitial fibrosis in T2DM, aggravating kidney disease and deteriorate kidney function thereby. The factorial analyzes show:T2DM and the chronic periodontitis had interacted synergy in DKD.
Chaper3Effect of Chronic periodontitis on expression of peroteins and their mRNA expression involved in renal lesions in OLETF rat
Objective
To detecte the changes of proteins and the corresponding mRNA expression of type IV collagen, and the expression of its four major regulators:TGF-β1, MMP-2, MMP-9, and TIMP-1of kidneys in the four groups for exploring whether chronic periodontitis affect the expression in the presence of T2DM. And To investigate the possible pathological mechanisms of the effects of chronic periodontitis on T2DM renal damage.
Methods
The expression of TGF-β1, MMP-2, MMP-9, TIMP-1, type IV collagen in the kidney tissue of four groups of rats were detected by immunohistochemistry ENVISION two-step semi-quantitative analysis. The rat serum concentration of TGF-β1was determined by ELISA assay. Meanwhile, using the SYBR Green real-time quantitative PCR (Real-Time PCR) technology (fluorescent dye incorporation) we tested type IV collagen, TGF-betal, MMP-2, MMP-9, TIMP-1, mRNA differences in the rats'kidney tissue. The experimental results analyzed by factorial design analysis of variance and two independent samples t-test.
Results
Type IV collagen was found in the glomerular basement membrane and mesangial matrix, blood vessel walls, tubular basement membrane etc. It was significantly higher in O group than that in the L group. The expression in O CP (+) group was significantly higher than that in the O CP (-) group.(T=11.099P=0.000<0.01). While there was no significant difference between the L CP (+) group and the L CP (-) group (t=2.032P=0.062).
TGF-β1was expressed in proximal convoluted tubule and distal convoluted and the expression was weak in renal glomerulus. It was found TGF-β1was positively expressed strongly in the kidneys of O CP (+) group. It's expression in O CP (+) group was significantly higher than CP (-) group (t=12958, P=0.000). It was significantly enhanced in L CP (+) group than that in L CP (-) expression (T=8.923, P=0.000). The expression significantly increased after the intervention of the CP. Group and periodontitis both has a significant impact on the expression of TGF-β1protein. Periodontitis and diabetes had corresponding interaction on the expression of TGF-betal protein (F=5.729P=0.024). TGF-β1mRNA expression trend is similar with and protein expression trend. Its expression increased significantly in L group after adding CP.
MMP-9protein was found mainly expressed in the proximal tubule and distal convoluted tubule epithelial cells and a small amount of expression was found in mesangial cells.In Normal control group L, the expression of MMP-9significantly higher than that of group O. the expression reduced significantly in L CP(+) group than in L CP (-)(t=11.274, P=11.274). There was an interactive effect between T2DM and periodontitis (F=66.397P=0.000). Renal MMP-9mRNA Expression of the overall trend in four groups was also agreed with the protein expression trend. In normal rat kidney of L group had the highest expression levels and the O group was significantly lower than that of group L.
MMP-2protein was expressed in the proximal tubule, distal convoluted tubule and glomerular visible trace. The little expression can also be found in the area of inflammatory infiltration of inflammatory cells. The highest expression level is still found in the L two groups, It was significantly decreased in L CP (+) group compared with L CP (-) group (t=9.030, P=0.000). O group was significantly reduced than in the L group. In O CP (+) group MMP-2positive staining area was not obvious. The main effect of group and periodontitis both had a significant impact on on expression of MMP-2protein. There is an interaction between group and periodontitis (F=13.529P=0.001). The MMP-2mRNA expression had the opposite trend in the four groups compared with its protein expression trend. It was significantly higher in group O than in group L. And it was significantly higher in O CP (+) group than that in O CP (-) group (t=2.475P=0.027).
The expression of TIMP-1protein was found in the proximal tubules, distal convoluted tubule, and renal interstitial and glomerular matrix region. The expression of TIMP-1in O group was significantly higher than that of group L (t=1721.404, P=0.000<0.01). The expression significantly enhanced in O CP (+) group than in the O CP (-) group (t=9.007P=0.000<0.01). The expression in L CP (+) group and L CP (-) group was close and found no statistically significant difference (t=1.245P=0.234).
The expression trend of TGF-beta1, and collagen type IV, TIMP-1mRNA in the four groups was consistent with the protein expression of respectively. Namely, L group have normal low expression of the above three gene. And the expression in O group was significantly increased than in L group (P<0.01). The mRNA expression in the O CP (+) group is slightly higher than the O CP (-) group without statistically difference.
Conclusion
In the presence of diabetes, periodontitis not only promoted fibrogenic core factor TGF-β1expression in kidney tissue but also increase TGF-β1serum level. In this combinding model, periodontitis also contributed to the high expression of TIMP-1which is the inhibition of collagen degradation enzymes. Futhermore, it inhibits extracellular mechanisms degrading enzymes MMP-2, MMP-9normal expression and thus led to an increase of type Ⅳ collagen deposition in kidney tissue.
This suggests the metabolic balance of extracellular matrix in the kidney has been broken with the increase of collagen production and the decreased collagen degradation in T2DM. This imbalance was exacerbated in the presence of periodontitis, resulting in the accelerated glomerulosclerosis and renal interstitial fibrosis. And finally the kidney function was impaired.
近年来,随着国内牙周医学的快速发展,越来越多的口腔医生并始关注牙周疾病,尤其是发病率极高的慢性牙周炎。美国人慢性牙周炎的发病率约47.6%,中国人群其发病率更高。众所周知,慢性牙周炎是菌斑微生物引起的慢性非特异性炎症性疾病,以造成牙周组织破坏、牙槽骨吸收和病理性牙周袋的行为为特征。牙周炎是一种炎症性疾病,但是我们不能再将其看作是一种局限于局部炎症,它可能通过增加机体全身炎症负担和增强机体免疫反应的途径来影响全身其它系统。
目前,有大量研究证明了牙周炎和全身各系统的疾病相关联。其中它与2型糖尿病(type2diabetes mellitus T2DM)的研究引起极大关注。T2DM是一种危害极大、发病率极高的慢性病。随着老龄化的进程,糖尿病合并牙周炎的患者急剧增加。现已证明,牙周炎与T2DM互为对方的独立危险因素。慢性牙周炎可加重机体全身炎症状态,升高全身炎症因子水平,促进胰岛素抵抗,不利于血糖控制,进而影响T2DM及其并发症的发生和发展。而T2DM时,机体对炎症的免疫力增强,牙周组织局部分泌过多的炎性细胞、炎性因子和蛋白酶,使得局部组织的破坏增加,加重牙周炎症程度。
而2型糖尿病其最大的危害在于其远期的慢性并发症,其中之一糖尿病肾病(Diabetic Kidney Disease DKD)是糖尿病患者的主要死亡原因之一。DKD的主要病理改变为肾脏细胞外基质-Ⅳ型胶原(胶原)的过度蓄积,产生肾小球硬化和肾间质纤维化。导致产生以上病变的机制复杂,但涉及几个关键的细胞因子和蛋白酶：转化生长因子β1(transforming growth factor--β1TGF-β1)在肾间质纤维化和肾小球硬化中起着关键作用；基质金属蛋白酶系统中MMP-2(明胶酶A),MMP-9(明胶酶B)以及抑制因子TIMP-1是研究发现主要调节肾脏细胞外基质(extracellular matrix ECM)平衡的关键因子。
目前对于牙周炎对T2DM的慢性并发症DKD影响的研究非常少,且多为临床病例的观察。因此本研究拟采用应用价值极高,且与人类T2DM发病极其相似的自发型2型糖尿病动物模型OLETF大鼠,在其上复合慢性牙周炎建立两种疾病复合动物模型的方法,排除其他混杂因素的干扰,初步探讨牙周炎对T2DM发生发展的作用。分析T2DM时牙周炎是否会对肾脏功能和肾脏组织结构产生影响。并通过检测与DKD发生密切相关的Ⅳ型胶原、TGF-β1、MMP-2, MMP-9, TIMP-1蛋白表达和基因转录水平的差异,进一步分析牙周炎对T2DM大鼠肾脏病损是否存在影响及其影响的病理机制,为进一步研究牙周炎与T2DM及DKD的相互关系建立可靠的实验基础,为临床多学科联合诊断,治疗和预防提供证据。
第一章OLETF大鼠慢性牙周炎模型的建立
目的
在OLETF大鼠到达糖耐量受损期时,通过牙周局部丝线结扎联合牙周局部涂菌建立糖尿病复合牙周炎模型,为下一步研究牙周炎对T2DM大鼠肾脏功能及肾脏病损是否存在影响及其影响的病理机制奠定基础。
方法
(1)自发性2型糖尿病OLETF大鼠(O组),4周龄,雄性,30只；同种系同周龄糖耐量正常的LETO大鼠(L组),雄性,20只。每4周行一次口服糖耐量检测,按2g/kg予50%葡萄糖溶液灌胃,依据OGTT试验的结果判定,血糖峰值>16.7mmol/L和葡萄糖灌胃后120min血糖>11.1mmol/L诊断为糖尿病,具备上述条件之一者为糖耐量受损(Impaired Glucose Tolerance IGT))。36周龄时,将到达糖耐量受损期(IGT期)OLETF大鼠26只,随即分为两组,一组附加牙周炎(O CP(+)组)(n=13),另外一组对照(O CP(-)组)(n=13)；LETO大鼠也随机分为两组,一组附加牙周炎(L CP(+)组)(n=10),另一组为正常对照(L CP(-)组)(n=10)。
(2)CP(+)组大鼠给予丝线分别结扎于左、右上颌第一磨牙(molar1, M1)及第二磨牙(molar2, M2)牙颈部,并在结扎处牙周涂抹四种牙周常见致病菌混悬液。定期检查丝线,有脱落者及时重新结扎并涂菌。
(3)每4周行一次OGTT,测体重,观察其体重和血糖的变化。并在36周龄和46周龄时,尾静脉采血测定其血清胰岛素水平的动态变化并计算糖尿病人胰岛素抵抗稳态模型(Homeostasis model assessment-insulin resistance HOMA-IR)值。
(4)56周龄时,处死所有实验大鼠,采集血清检测大鼠血脂水平；收集组织样本,颌骨样本采用Micro-CT扫描,EDTA脱钙后HE染色,观察牙周炎症状态。
四组之间的整体比较采用2×2析因设计资料的方差分析,两组间单因素数据比较采用独立样本t检验；同一处理因素两个不同水平之间以及同一水平不同处理因素之间的比较采用两独立样本的t检验。不同时间段多次测量的指标,总体各组和组内都用用重复测量的方差分析。同一时间同一组内有无干预的比较用两独立样本的t检验。
结果
本实验采用与人类T2DM发病及其相似的自发性T2DM大鼠OLETF大鼠,和其正常对照LETO大鼠作为研究对象。在36周龄时到达IGT期,模拟了人类T2DM的慢性发病的过程。
OLETF大鼠表现为多食多饮多尿,倦怠少动,体重快速增加。随着实验时间延长,体重差异进一步加大。O CP(+)组、O CP(-)组随着大鼠周龄的增加,血糖AUC显著增加有统计学意义(F=5.952P=0.029),但在O组任意周龄,O CP(+)组比O CP(-)组AUC略有增高,但并无显著差异。O组大鼠血清甘油三酯显著高于L组(t=11.324P=0.002),O CP(+)组比O CP(-)组甘油三酯水平有增高,并无显著差异(t=1.031P=0.320)。O组大鼠血清胆固醇显著高于L组(t=39.089P=0.000),O CP(+)组比O CP(-)组甘油三酯水平显著增高(t=2.532P=0.024),并且糖尿病和牙周炎二者之间存在交互作用(F=4.743P=0.038)
O组血清FI浓度和HOMA-IR值均显著高于L组。但O CP(-)组FI水平略高于O CP(+)组。O CP(-)组和O CP(+)组随着周龄增加HOMA-IR值的增加有显著性(F=38.233P=0.000<0.01),且同周龄时O CP(+)组比O CP(-)组HOMA-IR值高,但并无统计学差异。
牙周结扎大鼠口内检查发现牙颈部结扎处有食物残留,牙龈红肿、牙槽骨吸收、牙周袋形成,在O CP(+)组牙周炎的炎症状态更重；Micro-CT的三维重建显示四组大鼠牙槽骨吸收体积,O CP(+)组与L CP(+)组牙槽骨骨丧失量明显分别高于O CP(-)组和L CP(-)组,差别有统计学意义(P=0.013,P=0.004),且O CP(+)组骨丧失量要多于L CP(+)组骨丧失量。O组牙槽骨吸收体积总体上明显多于L组(t==11.476P=0.010)。
脱钙大鼠颌骨组织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。在OCP(+)组,炎症更重,可见结合上皮大幅根向迁移,牙周袋内及牙龈结缔组织内大量弥漫炎细胞浸润,甚至大量炎细胞游走到牙周袋内。
结论
OLETF大鼠在糖脂代谢和胰岛素抵抗方面可以较好的模拟人类T2DM的发病特征,是非常典型的T2DM动物模型。在OLETF大鼠附加牙周炎之后,牙周炎可促进胰岛素抵抗的程度和进度、不利于血糖控制,血清胰岛素代偿性增高,血脂增高,进而恶化T2DM的进程。T2DM也可加重牙周组织破坏,牙周炎症程度明显比单纯牙周炎组重。
第二章慢性牙周炎对大鼠肾功和肾脏组织形态的影响
目的
通过在OLETF大鼠附加牙周炎,探讨慢性牙周炎是否对OLETF肾脏功能和肾脏组织形态学存在影响。为探讨牙周炎是否影响T2DM并发症DKD的发生发展提供病理基础。
方法
收集大鼠处死时血清,全自动生化分析仪测大鼠血清反映肾功的生化指标,包括血清白蛋白(Albumin ALB)、总蛋白(Total protein TP)、尿素(Urea)肌酐(Creatinine Cr)四个指标。取单侧肾脏,肾门处纵剖肾脏组织常规制作组织2-3μm切片,分别行常规HE染色和PAS、MASSON、PASM特殊染色,结合起来全面分析肾脏病理改变。通过分析PAS染色切片计算肾小球硬化指数(glomerulosclerosis index GSI),利用MASSON染色计算肾间质纤维化指数(renal interstitial fibrosis index RIFI),比较各组间大鼠以上指标的差异。实验结果用析因设计的方差分析和两独立样本的t检验。
结果
O组比L组血清总蛋白和白蛋白水显著下降(t=11.131P=0.002)。一致的是O CP(+)组比O CP(-)组血清ALB和TP的水平均有显著下降。而在L组有无CP干预,二者之间的比较没有明显差异。O组大鼠血清肌酐水平均显著高于L组(t=9.099P=0.005)。L组大鼠有无CP干预肌酐水平无明显变化。O CP(+)组大鼠血清肌酐水平明显高于O组,差异有统计学意义(t=4.443P=0.000<0.01)。血清尿素结果显示O组比L组显著升高(P=0.024)。L CP(-)与L CP(+)组尿素水平接近。而O CP(+)组比O CP(-)尿素升高明显,但并无统计学差异。(t=1.484,P=-0.160)。
L CP㈩组组织学特征与L组接近,表现为正常肾脏的组织学特点。O组大鼠肾小球直径增大,鲍曼氏囊囊壁增厚明显,肾小球球囊部分粘连,可见囊腔扩大。肾小球系膜基质表现为中度至重度增生,呈弥漫性或结节状,再加上肾小球毛细血管基底膜增厚,部分区域出现局灶性肾小球硬化；肾小球周围出、入球小动脉管壁增厚。部分肾小管扩张,管腔内可见蛋白样物质或蛋白管型。肾小管上皮细胞玻璃样或空泡变性,部分区域甚至可见肾小管上皮萎缩、坏死、脱落,导致肾小管正常结构消失,晚期被纤维组织替代。间质可见散在炎细胞,严重者可见淋巴细胞和单核细胞成灶性浸润,肾间质纤维化。两种疾病的复合组病变程度更重。
O组大鼠肾GSI和RIFI均显著高于L组大鼠(t=85.112P=0.000<0.01,t=27887.6P=0.000<0.01)。在L CP(+)组,GSI略高于L CP(-)组,二者之间没有显著性差异(P=0.076)。OCP(+)组大鼠GSI与OCP(-)组比较有显著性差异(P值均<0.01)。而且T2DM和牙周炎对大鼠GSI存在交互效应(F=75.060,P=0.000<0.01)。RIFI也是相似的结果,在L CP(+)组,RIFI也略高于L CP(-)组,但二者之间没有显著性差异(P=0.296)。O CP(+)组大鼠与OCP(-)组比较有显著性差异(t=23.709P<0.01)。而且T2DM和牙周炎对大鼠RIFI存在交互效应(F=477.875,P=0.000)。
值得注意的是炎症浸润特征在O组很明显。在O CP(-)组8只大鼠组织切片中发现3只大鼠有大量散在炎细胞浸润或者大灶性炎细胞浸润(3/8),而在OCP㈩组中,有大量散在炎细胞浸润或者大灶性炎细胞浸润的大鼠例数明显增加,为6只(6/8),且炎细胞大多是单核细胞和巨噬细胞。
结论
本研究发现慢性牙周炎未对正常大鼠肾脏结构和功能产生显著影响；但在T2DM肾脏已有损害的情况下,慢性牙周炎显著增加了肾间质炎细胞浸润的风险,其促进了肾小球硬化、肾小管破坏及肾间质纤维化,加重了肾脏损害,恶化了肾功能,并且T2DM和牙周炎在肾脏病变发生中有交互、协同作用。
第三章慢性牙周炎对OLETF大鼠肾脏病变调控蛋白及其mRNA表达的影响
目的
检测各组大鼠肾脏组织中Ⅳ型胶原、TGF-β1、MMP-2, MMP-9, TIMP-1蛋白和基因转录水平的表达差异。分析牙周炎是否对肾脏组织代谢密切相关的关键物质的蛋白表达和基因转录水平产生了影响,进而探讨T2DM时慢性牙周炎影响肾脏损害可能的病理机制。
方法
采用免疫组化ENVISION二步法检测四组大鼠肾脏组织中Ⅳ型胶原、TGF-β1、MMP-2, MMP-9, TIMP-1蛋白的表达,进行半定量分析；ELISA法检测大鼠血清中TGF-β1的浓度；另采用实时定量PCR(Real-Time PCR)技术的Sybr green (荧光染料掺入)法来进行各组大鼠肾脏组织中Ⅳ型胶原、TGF-β1、MMP-2、MMP-9、TIMP-1、的mRNA基因表达差异分析。实验结果用析因设计的方差分析和两独立样本的t检验。
结果
IV型胶原蛋白表达于肾小球基底膜和系膜基质,血管壁、肾小管基底膜等部位。O组表达明显高于L组,且O CP(+)组与O CP(-)组之间表达量有统计学差异。(t=-11.099P=0.000<0.01),而L CP(+)组与L CP(-)组之间表达没有差异(t=2.032P=0.062)。
TGF-β1蛋白表达较广泛,主要集中在近曲小管上皮胞浆的部位,另外远曲小管上皮胞浆也有中等阳性的表达；肾小球基质和内皮的部位可见极少量表达。四组大鼠中TGF-β1均有表达,L组表达较低,O组比L组表达显著增强(t=2034.01P=0.000<0.01)。两组内在CP干预后其表达都明显增强。分组和牙周炎对TGF-β1蛋白的表达都有显著影响,并且牙周炎和糖尿病对TGF-β1蛋白的表达有交互相应(F=5.729P=0.024)。TGF-β1mRNA表达趋势与蛋白表达趋势一致。L组附加CP后其表达显著增高(t=4.851P=0.000<0.01)。
MMP-9蛋白主要表达于近曲小管和远曲小管上皮细胞,少量表达于系膜细胞。正常对照L组MMP-9的表达要明显高于O组(t=297.868P=0.000<0.01),L CP(+)组与L CP(-)组相比表达明显降低(t=11.274P=0.000<0.01)。O CP(-)组MMP-9蛋白表达的值与O CP㈩组接近,没有统计学差异(t=1.885,P=0.085)。T2DM和牙周炎之间有交互效应(F=66.397P--0.000)。四组大鼠肾脏MMP-9mRNA表达与其蛋白的表达总体趋势也一致,在正常L组大鼠肾脏都有高水平表达,而O组显著低于L组。但基因表达上O CP(+)组明显低于O CP(-)组(t=4.360P=0.001)。
MMP-2蛋白主要表达于近曲小管上皮细胞胞浆,远曲小管和肾小球可见微量表达,还可以发现在炎症浸润区域,炎细胞有少量表达。表达量最高的仍然是L两组,L CP㈩组与L CP(-)组相比表达明显降低(t=9.030P=0.000<0.01)。O组比L组显著降低(t=3184.226P=0.000<0.01)。O CP(+)组MMP-2蛋白表达值最低,阳性着色区域不明显。主效应分组和牙周炎对MMP-2蛋白的表达都有显著影响,主效应分组和牙周炎之间有交互效应(F=13.529P=0.001)。四组大鼠肾脏MMP-2mRNA表达与其蛋白的表达趋势正好相反。MMP-2基因表达O组显著高于L组(P<0.01),并且O CP(+)组的表达显著高于O CP(-)组(t=2.475P=0.027)。
TIMP-1蛋白表达于近曲小管、远曲小管、肾间质和肾小球基质区域。O组TIMP-1表达明显高于L组(t=1721.404P=0.000<0.01),O CP(+)组比O CP(-)组表达明显增强(t=9.007P=0.000<0.01),L CP㈩组与L CP(-)组相比表达接近,没有统计学差异(t=1.245P=0.234)。
四组大鼠肾脏TGF-β1, TIMP-1和IV型胶原mRNA表达与各自蛋白的表达趋势一致,即在L组大鼠肾脏都有正常的低水平表达,而以上三者在O组表达量比L组表达都显著上调(P<0.01),并且在O CP㈩组表达都略高于O CP(-)组,差异均没有统计学意义。
结论：
牙周炎复合糖尿病时,促进了肾脏组织以及血清中促纤维化的核心因子TGF-β1的表达增高,也促进了抑制胶原降解的酶TIMP-1的高表达；却抑制了细胞外机制降解酶MMP-2,MMP-9的正常表达,从而导致了肾脏组织IV型胶原沉积增多。
糖尿病状态下,肾脏细胞外基质的代谢平衡已经被打破,胶原生成增多,降解减少；而在糖尿病复合牙周炎后,这种不平衡更加恶化,牙周炎加剧了细胞外基质的代谢失衡,加速肾小球硬化和肾间质纤维化,促进肾脏功能的进一步丧失。
Background
In recent years, with the rapid progress of periodontal medicine, more dentists are beginning to pay much more attention to periodontal disease, especially the chronic periodontitis. Periodontitis is a common infection of the periodontal tissues and a major cause of tooth loss in adults. The most prevalent form is chronic periodontitis. The chronic periodontitis incidence rate in Americans is about47.6%in2010. Even the worse is the higher incidence in the Chinese population. As we all know, chronic periodontitis is a kind of non-specific inflammatory diseases caused by plaque microorganisms. That would result in periodontal tissue destruction, which is characterized by alveolar bone resorption and pathological periodontal pocket. Periodontitis is an inflammatory disease, but we would no longer regard it as a limited local inflammation. It might influence the whole body by the ways to increase the body's systemic inflammatory burden and enhance the immune response.
Currently, there are a large number of studies have revealed periodontitis and systemic disease associated, with type2diabetes (T2DM) is of great concern. T2DM is a kind of chronic diseases with high incidence and great harm. With the process of aging, diabetes and periodontitis patients have increased dramatically. It has been revealed that periodontitis and T2DM is the independent risk factor for each other. Chronic periodontitis may increase the body's systemic inflammatory burden, increased systemic levels of inflammatory factors, and promote insulin resistance. And that was not conducive to blood glucose control, thereby affecting the occurrence and development of T2DM and its complications. In T2DM, the enhancement of the immune system to the body inflammation leads to local excessive infiltration of inflammatory cells in periodontal tissues, excessive secretion of inflammatory factors and proteases. That would cause the more destruction in local tissue and increase the degree of periodontal inflammation.
The greatest hazards of T2DM are the long-term chronic complications. Diabetic kidney disease (DKD) is one of them and it is the leading cause of death of patients with T2DM. The main pathological changes of DKD are the renal extracellular matrix-type IV collagen (collagen) excessive accumulation, resulting in glomerulosclerosis and renal interstitial fibrosis. Mechanisms leading to the kidney lesions are very complex. Involving several key cytokines and proteases:TGF-β1plays a key role in renal interstitial fibrosis and glomerulosclerosis; matrix metalloproteinase MMP-2(gelatinase A), MMP-9(gelatinase B), and inhibitor TIMP-1were found to be the key factors to regulate renal ECM balance.
Until now, there are few researches on the effect of Periodontitis on the DKD. Only a few available studies were the observation of clinical cases. Therefore, this study intends to composite type2diabetes and chronic periodontitis disease in an animal model. We aimed to discusse the the role of periodontitis in the development of T2DM and to determine whether periodontitis impact renal function and renal structure by excluding the interference of any other confounding factors. And through testing the above mentioned DKD closely related type IV collagen, TGF-betal, MMP-2, MMP-9, TIMP-1protein and Mrna expression, we attempted to further analyze whether periodontitis had an impact on DKD and the possible pathological mechanisms. And try to establish a reliable experimental basis for further study of periodontitis and T2DM or DKD relationship, diagnosis, treatment and prevention for clinical Multidisciplinary combination therapy.
CHAPTER ONE The animal model of chronic periodontitis in OLETF rats
Objective:To establish experimental periodontitis in OLETF rats by ligature and periodontal pathogen infection for further evaluating the effects of periodontal inlammation on the diabetic kidney disease and the pathogenesis in OLETF rats.
Methods:
(1)30OLETF rats (O group),4-week-old, male, spontaneously type2diabetic;20LETO rats (L group), male, with the same germline and the same age but having normal glucose tolerance, as the control group. Every4weeks, oral glucose tolerance was determined with50%glucose solution2g/kg orally administered. Diabetes diagnosis is based on the results of the OGTT test, blood glucose peak>16.7mmol/L and glucose and blood glucose>11.1mmol/L in120min as the criteria. Meeting either one of the above criteria is recognized as impaired glucose tolerance (IGT). At36weeks old,(being IGT state)26OLETF rats were randomly divided into two groups:periodontitis group (O CP (+))(n=13) and non-periodontitis group (O CP (-) group)(n=13); LETO rats were also randomly divided into two groups, periodontitis group (L CP (+) group)(n=10) and non-periodontitis group (L CP (-) group)(n=10).
(2) The bilateral maxillary first and second molars in the [L CP (+)] group and [O CP (+)] group were ligatured with3/0silks soaked with periodontal pathogens, in subgingival position for28weeks. The ligatures were checked regularly and re-done if necessary.
(3) OGTT was conducted every four weeks and measured body weight at the same time. The rat tail vein blood samples were obtained at36weeks and46weeks of age to evaluate the dynamic changes of serum insulin levels and HOMA-IR score.
(4) At56weeks 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 periodontal tissue 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℃.
The overall comparisons between the four groups were done by using a2x2factorial design analyzes. Single factor data of the two groups were compared using independent samples t test. Independent samples t test were also used to compare the same factors at two different levels and the same level of different factors. Multiple indicators measured in different times between general groups or within groups were compared by by analysis of variance with repeated measurements. With or without the intervention of comparison in the same time within the same group was compared by the two independent samples t test.
Results
The spontaneous T2DM rats-OLETF rats and normal control LETO rats were used as research objects in this study. OLETF reached the IGT period at36weeks age vividly simulating human T2DM chronic disease process.
OLETF rats showed typical clinical features of the T2DM, just like polyphagia, polydipsia and polyuria, rapid weight gain. Blood glucose AUC of the OLETF rats increase statistically significant with ageing (F=5.952P=0.029). However, at any weeks of age, AUC slightly increased in O CP (+) group than in the O CP (-) group without significant difference. Serum triglyeride (TG) and cholesterol (CHOL) in O group were significantly higher than those in Group L. TG and CHOL in O CP (+) group were both higher than those in O CP (-) group, while serum CHOL difference with significance.
Serum FI and HOMA-IR in O group was significantly higher than that in Group L. O CP (-) group FI level was slightly higher than O CP (+) group. O CP (-) group and O CP (+) group had significantly increased HOMA-IR values with increasing age.
Intraoral Periodontal examination in CP (+) group show:food residue around tooth neck, swollen gums, alveolar bone resorption, periodontal pocket. O CP (+) group had heavier periodontal inflammation. Micro-CT three-dimensional reconstruction of the rat alveolar bone in four groups display that the alveolar bone loss in O CP (+) group and L CP (+) group were significantly higher than that in O CP (-) group and L CP (-) group, respectively. Moreover, the rats in O CP (+) group have more bone loss than L CP (+) group.
Through HE staining of decalcified rat mandible, we found periodontal attachment loss, destruction of collagen fibers, inflammatory cell infiltration in the end the periodontal pocket, alveolar bone resorption significantly. In the OCP (+) group, inflammation was more heavier, with junctional epithelium migrate to root direction, a large number of diffuse inflammatory cell infiltration in periodontal pockets and gingival connective tissue, and even migrate into the periodontal pocket.
Conculusion
OLETF rat is a very typical T2DM animal model which simulates human T2DM characteristics in glucose and lipid metabolism and insulin resistance vividly.
Periodontitis promote the degree and progress of insulin resistance in T2DM. It could deteriorate glycemic control and lead to compensatory high serum insulin. Thereby deteriorate the T2DM state. T2DM could aggravate periodontal tissue destruction conversly. The degree of periodontal inflammation in T2DM is much heavier than in normal periodontitis group.
Chaper2Effect of Chronic periodontitis on rat renal function and renal morphology
Objective
We aimed to investigate whether chronic periodontitis affect the OLETF kidney function and the morphology kidneys by measuring kidney function indices and observing the kidney morphology. To supply evidence for the Pathological basis of whether periodontitis affecting the development of T2DM complications.
Methods
The biochemical markers in the four rat groups including serum albumin (ALB), total protein (TP), urea (UREA) and creatinine (Cr) were analyzed by automatic biochemical analysis instruments. In order to do a comprehensive analysis of renal pathological changes, routine HE staining and PAS, MASSON, PASM special staining were adiministered. Glomerulosclerosis index (GSI) was calculated by analyzing the PAS stained sections. MASSON staining was used to calculate the index renal interstitial fibrosis (RIFI). Factorial design analysis of variance and two independent sample t-test were used for statistical analysis of the results.
Results
The levels of serum TP and ALB decreased significantly in O group than in the group L (t=11.131, P=0.002). Consistently, serum levels of ALB and TP decreased significantly in O CP (+) group than in O CP (-). In the L group or without CP intervention, there is no significant difference. Serum creatinine and urea levels were significantly higher in O gourps than in L groups (t=9.099, P=0.005).
The histological features in L CP (+) group were close to L group showing normal renal histological features. The glomerular diameter increased and cortex narrowed and medulla widened in OLETF rats. Bowman's capsule wall became thickened significantly, glomerular balloons were adhered partly and cysts increased. There are moderate to severe hyperplasia in glomerular mesangial matrix diffusively or nodularly, basement membrane thickening of glomerular capillary, focal glomerulosclerosis in some areas, glomerular capillary plexus lobulation, and vascular wall thickening of arteryies in and out of glomerular surrounding. There were hyaloid and vacuolar degeneration in cortical tubular epithelial cells, dilatation of some tubular, protein-like substances or protein casts in lumen. It was also found that there were tubular epithelial cell desquamation, tubular degeneration and atrophy in some areas, dilation or atrophy of hylic tubules, scattered or gathering infiltration of lymphocytes and mononuclear cells and fibrosis in interstitial areas.
Concerning the GSI and RIFI, their results are similar. The GSI and RIFI in OLETF rats are significantly higher than Leto rats (P<0.01). They are slightly higher in L CP (+) group than in L CP (-) group, no significant difference. However, they are significantly higher in OCP (+) rat than in (P<0.01). There is an interaction effect between T2DM and periodontitis.
It is noteworthy that obvious inflammatory infiltration was found in the O group. Among O CP (-) group, a large number of scattered inflammatory cell infiltration or large focal inflammatory cell infiltration were found in three of eight rats (3/8). In O CP (+) group, a large number of scattered inflammation cell infiltration or large focal inflammatory cell infiltration in rat patients was found in six of eight rats (6/8) and most of the inflammatory cells are monocytes and macrophages.
Conclusion
Chronic periodontitis may not cause significant changes in kidney function and histopathology in normal rats. However, chronic periodontitis may significantly increase the renal interstitial inflammatory cell infiltration risk in T2DM. Moreover, the chronic periodontitis promote kidney glomerulosclerosis and renal interstitial fibrosis in T2DM, aggravating kidney disease and deteriorate kidney function thereby. The factorial analyzes show:T2DM and the chronic periodontitis had interacted synergy in DKD.
Chaper3Effect of Chronic periodontitis on expression of peroteins and their mRNA expression involved in renal lesions in OLETF rat
Objective
To detecte the changes of proteins and the corresponding mRNA expression of type IV collagen, and the expression of its four major regulators:TGF-β1, MMP-2, MMP-9, and TIMP-1of kidneys in the four groups for exploring whether chronic periodontitis affect the expression in the presence of T2DM. And To investigate the possible pathological mechanisms of the effects of chronic periodontitis on T2DM renal damage.
Methods
The expression of TGF-β1, MMP-2, MMP-9, TIMP-1, type IV collagen in the kidney tissue of four groups of rats were detected by immunohistochemistry ENVISION two-step semi-quantitative analysis. The rat serum concentration of TGF-β1was determined by ELISA assay. Meanwhile, using the SYBR Green real-time quantitative PCR (Real-Time PCR) technology (fluorescent dye incorporation) we tested type IV collagen, TGF-betal, MMP-2, MMP-9, TIMP-1, mRNA differences in the rats'kidney tissue. The experimental results analyzed by factorial design analysis of variance and two independent samples t-test.
Results
Type IV collagen was found in the glomerular basement membrane and mesangial matrix, blood vessel walls, tubular basement membrane etc. It was significantly higher in O group than that in the L group. The expression in O CP (+) group was significantly higher than that in the O CP (-) group.(T=11.099P=0.000<0.01). While there was no significant difference between the L CP (+) group and the L CP (-) group (t=2.032P=0.062).
TGF-β1was expressed in proximal convoluted tubule and distal convoluted and the expression was weak in renal glomerulus. It was found TGF-β1was positively expressed strongly in the kidneys of O CP (+) group. It's expression in O CP (+) group was significantly higher than CP (-) group (t=12958, P=0.000). It was significantly enhanced in L CP (+) group than that in L CP (-) expression (T=8.923, P=0.000). The expression significantly increased after the intervention of the CP. Group and periodontitis both has a significant impact on the expression of TGF-β1protein. Periodontitis and diabetes had corresponding interaction on the expression of TGF-betal protein (F=5.729P=0.024). TGF-β1mRNA expression trend is similar with and protein expression trend. Its expression increased significantly in L group after adding CP.
MMP-9protein was found mainly expressed in the proximal tubule and distal convoluted tubule epithelial cells and a small amount of expression was found in mesangial cells.In Normal control group L, the expression of MMP-9significantly higher than that of group O. the expression reduced significantly in L CP(+) group than in L CP (-)(t=11.274, P=11.274). There was an interactive effect between T2DM and periodontitis (F=66.397P=0.000). Renal MMP-9mRNA Expression of the overall trend in four groups was also agreed with the protein expression trend. In normal rat kidney of L group had the highest expression levels and the O group was significantly lower than that of group L.
MMP-2protein was expressed in the proximal tubule, distal convoluted tubule and glomerular visible trace. The little expression can also be found in the area of inflammatory infiltration of inflammatory cells. The highest expression level is still found in the L two groups, It was significantly decreased in L CP (+) group compared with L CP (-) group (t=9.030, P=0.000). O group was significantly reduced than in the L group. In O CP (+) group MMP-2positive staining area was not obvious. The main effect of group and periodontitis both had a significant impact on on expression of MMP-2protein. There is an interaction between group and periodontitis (F=13.529P=0.001). The MMP-2mRNA expression had the opposite trend in the four groups compared with its protein expression trend. It was significantly higher in group O than in group L. And it was significantly higher in O CP (+) group than that in O CP (-) group (t=2.475P=0.027).
The expression of TIMP-1protein was found in the proximal tubules, distal convoluted tubule, and renal interstitial and glomerular matrix region. The expression of TIMP-1in O group was significantly higher than that of group L (t=1721.404, P=0.000<0.01). The expression significantly enhanced in O CP (+) group than in the O CP (-) group (t=9.007P=0.000<0.01). The expression in L CP (+) group and L CP (-) group was close and found no statistically significant difference (t=1.245P=0.234).
The expression trend of TGF-beta1, and collagen type IV, TIMP-1mRNA in the four groups was consistent with the protein expression of respectively. Namely, L group have normal low expression of the above three gene. And the expression in O group was significantly increased than in L group (P<0.01). The mRNA expression in the O CP (+) group is slightly higher than the O CP (-) group without statistically difference.
Conclusion
In the presence of diabetes, periodontitis not only promoted fibrogenic core factor TGF-β1expression in kidney tissue but also increase TGF-β1serum level. In this combinding model, periodontitis also contributed to the high expression of TIMP-1which is the inhibition of collagen degradation enzymes. Futhermore, it inhibits extracellular mechanisms degrading enzymes MMP-2, MMP-9normal expression and thus led to an increase of type Ⅳ collagen deposition in kidney tissue.
This suggests the metabolic balance of extracellular matrix in the kidney has been broken with the increase of collagen production and the decreased collagen degradation in T2DM. This imbalance was exacerbated in the presence of periodontitis, resulting in the accelerated glomerulosclerosis and renal interstitial fibrosis. And finally the kidney function was impaired.
引文
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