PPARγ在牙龈卟啉单胞菌引起内皮功能紊乱中的作用及机制

Periodontal pathogen was closely related to endothelial dysfunction, Nitric oxide (NO) as vascular diastolic substance play an important in endothelial dysfunction. Porphyromonas gingivalis (Pg) can stimulate the release of NO in endothelial cells. Peroxisome proliferator-activated receptor gamma (PPARγ) is a transcriptional factor, the effect and mechanism of PPARγ involved in NO synthesis induced by Pg in endothelial cells is still uncleared. It is important to make clear of that not only for prevention and treatment of periodontal disease but also for atherosclerosis. To explore that, this study intend to build a Pg-invasion-endothelial-cell-model. The level of PPARγ mRNA and protein will be analysed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. PPARγ transcriptional activity will be observed through the detection of luciferase reporter gene plasmid. Cellular reactive oxygen species level will be detected through the DCFA-AM fluorescent probe and enzyme-linked immunosorbent assay(ELISA) technique , then tetrahydrobiopterin and NO content also analysed by ELISA when PPARγwas activated by its ligand 15d-PGJ2 or blocked by inhibitor GW9662. By using PPARγligand 15d-PGJ2 , inhibitor GW9662, phosphatidylinositol 3 kinase(PI3K) inhibitor(wortmannin), endothelial nitric oxide synthases(eNOS)inhibitor (Cavtratin),extracellular signal-regulated kinases (ERK) inhibitor (PD98059), C-Jun N-terminal kinases(JNK) inhibitor (SP600125), p38 mitogen activated protein kinase(MAPK) inhibitor (SB203580), nuclear factor-κB (NF-κB) inhibitor (SN50), inducible nitric oxide synthases（iNOS） inhibitor (1400W) the exploration of PPARγ's ability to modulate the PI3K/Akt/eNOS signaling pathway and MAPK/NF-κB/iNOS signaling pathway will be researched. This project will clarify the molecular mechanisms of PPARγ involved in endothelial dysfunction induced by Pg, will provide a new target on improving endothelial dysfunction induced by Pg.

牙周致病菌和内皮功能紊乱密切相关。牙龈卟啉单胞菌Pg侵入内皮细胞时，转录因子PPARγ的表达及其影响血管舒张物质NO合成的具体分子机制还不清楚，明确此机制，对防治牙周病和动脉粥样硬化均具有重大意义。本研究拟通过建立Pg侵入人脐静脉内皮细胞的模型，应用RT-PCR和Western blot 检测PPARγmRNA和蛋白表达的改变，通过荧光素酶报告基因质粒检测PPARγ转录活性变化；通过DCFA-AM荧光探针等技术检测细胞活性氧簇（ROS）的改变，并分析PPARγ被配体激活或阻断剂作用后细胞ROS水平的改变，进而对四氢生物蝶呤和NO合成量的影响；通过加入特异的阻断剂和配体，探索PPARγ对PI3K/Akt/eNOS和MAPK/NF-κB /iNOS信号通路的影响，以期明确Pg导致内皮功能紊乱中PPARγ影响NO合成的具体分子机制，并为改善Pg所致内皮功能紊乱治疗寻找新靶点。

牙龈卟啉单胞菌（Porphyromonas gingivalis, P.gingivalis）侵入时，转录因子过氧化物酶体增生物激活受体（peroxisome proliferator-activated receptor, PPAR）γ的表达及其影响血管收缩物质一氧化氮合成的具体分子机制还不清楚，明确此机制，对防治牙周病和动脉粥样硬化均具有重大意义。本研究成功建立P.gingivalis侵入人脐静脉血管内皮细胞系EA.hy926的模型。应用realtime PCR检测PPARγmRNA表达在Pg刺激后1小时后显著升高，应用Western blot检测PPARγ蛋白表达在Pg刺激后2小时显著升高，通过荧光素酶报告基因质粒检测PPARγ转录活性亦显示在Pg刺激后PPARγ转录活性显著增强，证实PPARγ参与了P.gingivalis刺激血管内皮细胞的过程。后续研究设计了四组，分别是对照组为EA.hy926加培养基，P.gingivalis刺激组为P.gingivalis W83刺激EA.hy926细胞，PPARγ激活组为提前30分钟加入PPARγ的激动剂15d-PGJ2预处理后再用P.gingivalis 刺激细胞，PPARγ抑制组为提前30分钟加入PPARγ的拮抗剂GW9662预处理后再用P.gingivalis 刺激细胞。研究结果显示在P. gingivalis刺激组细胞内的活性氧水平和抗氧化应激的产物均显著高于对照组。NO合成量在P.gingivalis刺激组4小时达到峰值，在PPARγ激活组P.gingivalis刺激8小时候达到峰值。Western blot检测结果显示P.gingivalis刺激时iNOS蛋白表达增加，eNOS蛋白表达下降，PPARγ通过PI3K/Akt通路增加p-eNOS表达从而增加NO产量。P.gingivalis刺激时， ERK蛋白表达显著下降，PPARγ激活后ERK蛋白表达升高，提示PPARγ亦通过ERK通路影响NO产量。综上PPARγ参与了Pg刺激血管内皮细胞过程，PPARγ激活可通过PI3K/Akt/eNOS及ERK通路影响NO合成，PPARγ配体有望改善P.gingivalis引起的内皮功能紊乱。