Nrf2对高脂饮食诱导肥胖引起的免疫紊乱的调节机制的探讨

Obesity has become one of the major factors that effect human’s health. It has been demonstrated that Nrf2 plays negative roles in regulating high-fat diet induced-obesity. After one dose of nonlethal administration of LPS, Nrf2-KO mice exhibited more severe pulmonary inflammation and dramatically increased the mortality, which suggested that the deficiency of Nrf2 will induce immunity disorder. In order to determine Nrf2 function in the regulation of immunity response to high-fat diet induced-obesity, Nrf2-KO and Keap1-KD (overexpressed Nrf2) mice will be used and induced to obesity by high-fat diet for 6 or 24 weeks, the related inflammation cytokines and the ratio of T CD4+/CD8+ lymphocyte will been measured. In addition, primary T cells from thymus wiil be isolated and treated with free fatty acid of palmitate, the secreted cytokines and the ratio of T cells will be measured. Also, the stable cell line of Si-Nrf2 and Si-Keap1 will be generated on CTLL-2 cell, then cells will be treated with free fatty acid of palmitate, try to confirm the observation from animals and primary hepatocytes experiments. Finally, it will be determined whether Nrf2 will regulate PTP-PTP gene expression by changing the binding activity with ARE binding site in the promoter, or change the phosphorylation status which regultated by changing PI3K acitivity, or regulate ROS status in the cells and modulate PTP-PEP activity, that regulate TCR activation, finally attenuates immunity activation, that avoid of the over activation of immunity response.

Nrf2对高脂饮食诱导的肥胖具有抑制作用。在LPS引起的急性炎症模型中，Nrf2-KO小鼠表现出更严重的肺部炎症和更高死亡率，显示Nrf2缺失加剧免疫紊乱。为明确Nrf2蛋白在由于高脂饮食引起的肥胖而导致的免疫紊乱过程中的调节作用，本项目拟使用Nrf2-KO和Keap1-KD小鼠，高脂饮食诱导6周和24周导致肥胖，检测血液中炎症因子和淋巴细胞种类和比例的变化。分离小鼠胸腺T淋巴细胞，使用自由脂肪酸诱导，检测TNF、IL2和IL6的表达变化，以及T细胞CD4+/CD8+比例变化。构建Si-Nrf2和Si-Keap1细胞系，明确Nrf2对细胞分泌炎症因子的影响，并检测免疫相关基因的表达变化。进一步证明Nrf2是否通过ARE结合位点调节PTP-PEP表达，是否通过调节ROS影响PTP-PEP活性，是否通过PI3K影响PTP-PEP磷酸化水平，调节TCR活性，导致免疫抑制，避免机体的免疫过度激活。

在正常条件下，Nuclear factor erythoid 2-related factor 2 （Nrf2）与Kelch-like ECH-associated protein 1（Keap1)蛋白相结合，受Keap1介导的蛋白酶体系统降解；当机体或细胞受内外界氧化或化学等刺激，Keap1构象发生改变或Nrf2直接被磷酸化,导致Nrf2与Keap1解离，Nrf2蛋白不再受蛋白酶体降解系统降解，在细胞胞浆中聚集，进入细胞核内，与抗氧化反应元件(Antioxidant Response Element, ARE)结合,启动下游解毒酶、抗氧化蛋白等基因表达，抵抗有害刺激。而有关Nrf2对脂肪和糖代谢调节，进而影响肥胖发生和糖尿病病程进程还鲜见报道。本项目通过制作Nrf2和Leptin双基因敲除小鼠，研究Nrf2基因缺失对脂肪代谢和肥胖发生的调节作用，以及对诱发的糖尿病病症的影响，为探讨Nrf2激动剂或抑制剂在阻止肥胖发生，或者治疗肥胖等实际应用中提供有力依据。Nrf2基因缺失促进正常小鼠白色脂肪组织堆积，抑制肥胖小鼠白色脂肪形成，并抑制肥胖小鼠脂肪组织中与脂肪发生（Adipogenesis）和脂肪合成（Lipogenesis）相关基因表达。另外，Nrf2基因缺失可促进正常小鼠肝脏细胞中脂肪堆积，却抑制肥胖小鼠肝脏细胞内脂肪堆积，其机制可能是降低与脂肪合成密切相关的转录因子Pparγ转录表达，降低NADPH含量，从而抑制肝脏细胞中脂肪合成，以及提高肝脏细胞中极低密度脂蛋白（VLDL）释放；显示Nrf2对脂肪组织和肝脏组织中的脂肪代谢的影响受Leptin基因信号通路调控。在正常和肥胖小鼠中，Nrf2基因缺失都引起葡萄糖耐受能力减弱，延长了急性葡萄糖实验中的高血糖症，其可能分子机理是降低了胰岛素诱导的Akt磷酸化水平增加。Nrf2基因缺陷导致OB-Nrf2KO小鼠血液甘油三酯含量显著升高，高密度脂蛋白胆固醇含量降低，Apolipoprotein B基因表达增加，引起肥胖小鼠血脂代谢障碍。本研究显示Nrf2对脂肪的发生和合成至关重要，Nrf2基因缺失可阻止肥胖小鼠肥胖病症和脂肪肝进一步恶化，但降低了胰岛素信号，引起了血脂代谢异常。推测在OB-Nrf2KO小鼠中，出现更为严重的炎症反应或免疫紊乱，或者说脂肪发生和脂肪合成的抑制是以机体代谢紊乱为代价，有关该方面研究还在继续探讨中