核受体NR2E1通过SIRT1/NF-κB/RelA通路调控肥胖相关脂肪组织慢性炎症的机制研究

Chronic inflammation of adipose tissue is an important factor leading to metabolic disorders. Attenuating chronic inflammation of adipose tissue plays an important role in preventing obesity-related metabolic diseases. NR2E1 involves in many cellular biological processes. Our preliminary study has found that NR2E1 expression showed a compensatory increase in PBMC of obese people, and was correlated with increased secretion of TNF-α and Il-6. NR2E1 in 3T3-L1 adipocytes could inhibit the secretion of TNF-α and Il-6 stimulated by palmitic acid. Microarray analysis suggested that NR2E1 could up-regulate the expression of SIRT1 and inhibit the inflammatory pathways. To this end, we propose the hypothesis: NR2E1 regulates obesity-related chronic inflammation of adipose tissue through SIRT1/NF-κB/RelA pathway. In order to test the hypothesis, we intend to detect the expression of NR2E1 and the change of downstream pathway under high-fat condition at cell and animal levels. Then we will interfere the expression of NR2E1 by lentivirus transfection, siRNA and gene knockout mouse model, and detect the expression and interaction of key signal molecules using PCR, Western Blot, Luciferase reporter assay, ChIP and immunofluorescence staining. Our research will elucidate the molecular mechanism of the regulating role of NR2E1, providing strategies for attenuating chronic inflammation of adipose tissue and preventing obesity-related diseases.

脂肪组织慢性炎症是导致代谢紊乱的重要因素，改善脂肪组织慢性炎症对阻止肥胖引发代谢性疾病有重要的意义。NR2E1参与调控细胞多种生物学过程。我们前期研究发现NR2E1与肥胖患者慢性炎症状态密切相关；在3T3-L1脂肪细胞中能够抑制棕榈酸介导的TNF-α、IL-6分泌；基因芯片分析提示NR2E1能够上调SIRT1表达并抑制炎症反应通路。据此，我们提出假说：NR2E1通过SIRT1/NF-κB/RelA通路调控肥胖相关脂肪组织慢性炎症。为验证假说，我们拟在细胞和动物水平检测高脂状态下NR2E1及下游信号通路的变化；通过慢病毒转染、siRNA及构建基因敲除鼠模型干预NR2E1表达，PCR、Western Blot、免疫荧光、Luciferase reporter assay、ChIP等手段检测信号关键因子的表达和相互作用，阐明其分子机制，为改善脂肪组织慢性炎症及肥胖相关疾病的防治提供策。

脂肪组织慢性炎症是导致代谢紊乱的重要因素，改善脂肪组织慢性炎症对阻止肥胖引发代谢性疾病有重要的意义。为探讨NR2E1在脂肪组织胰岛素抵抗和慢性炎症中的作用，我们建立了肥胖模型小鼠和NR2E1基因敲除小鼠。我们发现高脂喂养的肥胖小鼠NR2E1表达上调，NR2E1基因的缺失能够加重肥胖所致的脂质代谢的紊乱和慢性炎症。NR2E1基因敲除小鼠空腹血糖浓度更高，空腹胰岛素水平更低，葡萄糖耐量受损更严重，提示NR2E1基因的缺失加重肥胖导致的胰岛素抵抗。为了进一步探讨NR2E1基因缺失加重肥胖导致胰岛素抵抗的分子机制，我们通过western blotting检测了胰岛素相关的IRS1-PI3K-AKT信号通路。我们发现NR2E1基因敲除小鼠的IRS1和AKT磷酸化水平低于野生型小鼠，表明胰岛素信号通路受到抑制，此外，NR2E1基因敲除组GLUT4的表达也较对照组显著降低，在高脂饮食喂养16周后，该信号通路抑制得更为明显，提示胰岛素抵抗更为严重。NR2E1基因敲除小鼠胰岛素敏感性受损可能是由于下游胰岛素信号通路受到抑制所致。在肥胖及超重人群中，我们发现NR2E1的水平明显高于BMI正常的人群。在肥胖及超重人群中NR2E1 mRNA的水平和TC、FFA, IL-6, TNF-α 和 LDL-c呈正相关。在3T3-L1脂肪细胞中过表达NR2E1会增加SIRT1同时减少acetyl-NF-κB p65, TNF-α and IL-6的表达，提示NR2E1可能通过SIRT1/ NF-κB p65通路负向调节3T3-L1脂肪细胞的炎症过程。综上所述，我们的研究分别从动物、细胞、临床实验等层面揭示了NR2E1在肥胖所导致的代谢紊乱和慢性炎症中的重要调节作用，提示NR2E1可能是调节肥胖相关慢性炎症的重要潜在治疗靶点。