牙周微生物多样性调控TLRs信号通路在IgA肾病中的作用及机制研究

IgA nephropathy (IgAN) has obvious racial and regional differences of incidence. The study found that, the incidence of IgAN in Xinjiang periodontitis patient was significantly higher than that in non periodontitis, the prevalence rate of periodontitis and the incidence of IgAN were significantly higher in Uygur populations than the local populations.We also observed from the zoopery that, periodontitis can promote the progression of the IgAN, And periodontitis leads to inflammatory reaction. Therefore we put forward a hypothesis that: periodontitis stimulates the host cells to release inflammatory factors through the activation of TLRs signal pathway, and then causes the immune inflammatory reaction, to promote the progression of IgAN. In order to verify this hypothesis, we intend to use high-throughput sequencing technologies to analysis the diversity of oral microorganisms in Uighur populations, screening of dominant bacteries, on the basis of this, to establish chronic periodontitis and IgAN model of a rat, examined the protein expression level of inflammatory factors and the levels of the TLR4/9 receptors; Isolated and cultured rat mesangial cell, blocking the cell's TLR4/9 signaling pathway, to detect the inflammatory factor’s expressions in mesangial cells and the level of mRNA signaling pathway at the before and after of the regulation, to clear the correlations and mechanisms of the chronic periodontitis and IgAN. This study will lay the theoretical foundation for IgAN patients in Xinjiang Uygur populations with early intervention of periodontal treatment, at the same time will also provide new evidences for the IgAN Mucosal pathogens theory.

IgA肾病(IgAN)发病率存在明显种族与地域差异，研究发现新疆慢性牙周炎患者IgAN发病率显著高于非牙周炎，维吾尔人群牙周炎及IgAN患病率均高于当地人群，我们动物实验也观察到牙周炎可促进IgAN病变进展，与牙周炎引发机体炎性反应有关。因此我们提出假说：牙周炎通过激活TLRs信号通路，刺激宿主细胞释放炎症因子，进而引起免疫炎症反应，促进IgAN发生进展。为验证这一假说，我们拟采用高通量测序技术分析维吾尔人群口腔微生物多样性，筛选优势菌群，并据此建立大鼠慢性牙周炎及IgAN模型，检测炎症因子蛋白表达水平及TLR4/9受体水平；分离培养大鼠系膜细胞，阻断细胞TLR4/9信号通路，检测调控前后系膜细胞炎症因子表达及信号通路mRNA水平，明确慢性牙周炎与IgAN的相关性及作用机制。本研究将为新疆维吾尔IgAN患者牙周早期干预治疗奠定理论基础，同时也将为IgAN粘膜致病菌学说提供新的依据。

新疆地区维吾尔族人群IgA肾病患病率明显高于汉族人群，而牙周炎的患病率明显高于其他地区，既往研究提示牙周炎可能促进IgA肾病的发生与发展，其中牙周致病厌氧菌的LPS（Lipopolysaccharide脂多糖）能与牙周膜表面TLR4、TLR9结合，激活NF-κB，产生一系列炎症因子促进牙周组织破坏，体外实验证实IgA肾病与TLRs有关。课题组采用高通量测序技术分析维吾尔人群口腔微生物多样性，筛选优势菌群；建立大鼠慢性牙周炎及IgAN模型，检测各组大鼠口腔外周血及牙周、肾组织标本中TNF-α、IL-6、IL-1β等相关炎症因子表达水平及组织中TLR4、TLR9、NF-κB水平；分离大鼠肾脏系膜细胞并体外培养，阻断系膜细胞TLR4/9信号通路，检测阻断前后细胞炎症因子表达水平及TLR4、TLR9 受体和核转录因子NF-κB的mRNA水平，明确慢性牙周炎与IgAN的相关性及作用机制。我们的研究表明牙周炎+IgA肾病组患者唾液较牙周炎组在属水平上Neisseria显著升高、Tannerella显著降低；较IgA肾病组在属水平上Porphyromonas、Haemophilus显著升高。牙周炎+IgA组大鼠牙周及肾脏组织中TLR4、TLR9 mRNA、蛋白均高于IgA、牙周炎、对照组，且牙周炎+IgA组NF-κB p-p65显著高于牙周炎组（0.827±0.031，0.699±0.038）。牙周炎+IgA组较其他组牙周及肾脏病理改变重。体外培养肾小球系膜细胞发现TLR4抑制剂能够显著抑制因LPS升高的TLR4和NF-κB mRNA（2.475±0.289 ，0.610±0.132；3.277±0.331，2.208±0.619），以及TLR4、TLR9、NF-κB p-p65蛋白（0.770±0.039，0.597±0.019；0.744±0.015，0.533±0.024；0.698±0.029，0.541±0.039）。TLR4、TLR9抑制剂可抑制体外培养的肾小球系膜细胞上清液中因是LPS升高的IL-1β、IL-6、TNF-α。因此我们得出结论：牙周致病菌可以通过TLR4/9激活NF-κB，释放炎症因子，加重牙周炎及IgA肾病，为IgA肾病的诊断治疗提供新的临床及基础研究方向。