ccf-mtDNA诱导小胶质细胞炎症反应及其影响衰老和肥胖的研究

Hypothalamic microglial IKK-β/NF-κB plays an important role in regulating energy homeostasis; however, the causes and characteristics remain unclear. Here, using microrglial cells in vitro, we found that the increase in the levels of circulating cell-free mtDNA (ccf-mtDNA) may serve as a trigger of colony-stimulating factors (CSFs) expression through NF-κB activation in microglial cells. Further, we found that the production of hypothalamic CSFs and ccf-mtDNA content in cerebrospinal fluid is excessive under conditions of aging or obesity, which are both associated with the dysregulation of energy homeostasis. Central administration of CSFs or mtDNA to adult mice caused metabolic dysfunction, including glucose intolerance and significant changes in food intake, body weight. In summary, these results led us to predict that ccf-mtDNA -induced CSFs excess via activation of hypothalamic microglial IKK-β/NF-κB in aging or obesity and CSFs seem to be involved in IKK-β/NF-κB-mediated microglia–neuron crosstalk that controls systemic ageing and obesity. Some mouse model with IKK-β knockout or constitutively active in the microglia of mediobasal hypothalamus, or with CSF receptors knockdown of pro-opiomelanocortin (POMC) or agouti related protein (AgRP) neuron will develop to reveal a microglia–neuron crosstalk via microglial IKK-β/NF-κB and the bi-directional effects of hypothalamic microglial IKK-β/NF-κB, which will have broad implications for the causes and characteristics of hypothalamic microglial IKK-β/NF-κB inflammatory program in obesity and aging. It may represent potential strategies for combating ageing- or obesity-related health problems.

下丘脑小胶质细胞的NF-κB通路在衰老或肥胖的发生发展中尤为重要，但是它的激活机制以及对代谢的影响仍然知之甚少。我们通过mtDNA等刺激多种神经细胞，发现mtDNA可以灵敏快速地激活NF-κB来促使集落刺激因子表达增高；在衰老和肥胖小鼠中发现其脑脊液ccf-mtDNA和下丘脑集落刺激因子水平升高；第三脑室注射mtDNA或集落刺激因子可以引起代谢紊乱。我们推测小胶质细胞外游离的mtDNA增多可以通过激活NF-κB通路促使集落刺激因子分泌进而调控下丘脑代谢相关神经元。我们将利用多种关于下丘脑的小鼠模型（小胶质细胞IKK-β敲除和激活、POMC等神经元表面集落刺激因子受体敲低），来明确小胶质细胞NF-κB通路、ccf-mtDNA、集落刺激因子和下丘脑代谢相关神经元之间的关系，并系统性地阐明下丘脑小胶质细胞NF-κB通路在衰老或肥胖发生发展中的机制和作用，为延缓衰老或改善肥胖提供新的理论研究基础。

现今，衰老和肥胖无疑已经成为日益严重的全球公共健康问题。两者存在很多共性，特别是引起首要原因都是能量代谢失衡。其中，下丘脑小胶质细胞的NF-κB通路在衰老或肥胖的发生发展中尤为重要，但是它的激活机制以及对代谢的影响仍然知之甚少。本研究共五部分：首先建立了脑脊液和细胞培养上清液中循环游离线粒体DNA（ccf-mtDNA）拷贝数分析的绝对定量方法；通过同位素标记相对和绝对定量技术对初衰和肥胖小鼠大脑进行蛋白组学分析，初步筛选出大脑衰老初期和全身性肥胖进程中与线粒体和集落刺激因子相关的共同差异表达基因共14个；通过体外实验发现小胶质细胞外游离的mtDNA增多可以灵敏地激活NF-κB通路从而引起小胶质细胞炎症反应并促使小胶质细胞集落刺激因子表达增高，而小胶质细胞外M-CSF增多又可进一步促进小胶质细胞外游离的mtDNA增多，G-CSF和GM-CSF增多反而促进小胶质细胞外游离的mtDNA减少；通过比较28~30月龄小鼠和3月龄小鼠以及不同时间段的高脂饮食小鼠和常规饮食小鼠，发现衰老和12周HFD引起的肥胖会导致脑脊液中cf-mtDNA水平和下丘脑中集落刺激因子表达升高。通过第三脑室注射，发现M-CSF会引起小鼠摄食量增加、体重增加和葡萄糖耐量受损，G-CSF、GM-CSF和mtDNA可以引起小鼠摄食量减少、体重减少和葡萄糖耐量受损；最后通过慢病毒转染技术和Cre-lox或Cre-STOP-lox系统建立下丘脑小胶质细胞IKK-β敲除和激活小鼠模型，进一步阐明下丘脑小胶质细胞NF-κB通路对衰老和肥胖的影响。本研究揭示了小胶质细胞NF-κB 通路介导的cf-mtDNA/集落刺激因子相互作用机制，并首次详细阐述了衰老和肥胖时下丘脑小胶质细胞NF-κB通路对能量代谢的双向调节作用，有望为进一步揭示小胶质细胞激活的原因和作用提供更广阔的研究方向，为延缓衰老或改善肥胖提供新的理论研究基础。