脂联素中枢成骨作用及其机制研究

Adiponectin (APN) is an adipose tissue-derived hormone with antidiabetic， insulin-sensitizing, regulating appetite and energy balance properties.The ventromedial hypothalamic nucleus (VMH）region in hypothalamus has been demonstrated promote bone resorption through upregulating osteoblast RANKL expression and inducing osteocalst proliferation and differentiation via enhancing sympathetic tone. A few studies showed that a communicative link exists between APN and the brain, specifically in the hypothalamic region. APN treatment increased energy expenditure (EE), decreased body weight, glucose homeostasis and depressant-like activity. However, whether APN acts in the brain to regulate bone formation is unknown. In this study, we aimed to determine the effects and mechanisms of APN in the hypothalamus on bone formation. We applied long-term intracerebroventricular (ICV) infusion of globular APN (gAPN) and full-length APN (fAPN) into APN-deficient (APN-/-) and wild type (WT) mice and also used APPL1-shRNA lentivirus to specifically block APPL1 to measure APN signaling and APN-mediated downstream effects (bone formation, sympathetic tone, and energy consumption). To explore the APN signaling pathways in hypothalamus, we performed real-time PCR, western blot and immunohistochemistry analysis with an hypothalamus cell line mHypoE-N1 in vitro with differet doses of APN (0.1, 1, 10 nM). Based on all this designed experimetns and according data, the present study will provide a basis of APN promoting bone formation through a hypothalamus relay and the related information underlying signal transduction pathway.

脂联素（adiponectin）是脂肪组织分泌的具有抗糖尿病和促胰岛素增敏、调控食欲和能量代谢等作用的激素。以往的研究表明下丘脑腹内侧核（ventromedial hypothalamic nucleus， VMH）通过激活下游交感神经，上调成骨细胞RANKL表达，诱导破骨细胞分化和增殖，进而促进骨吸收。近来研究发现脂联素可作用于下丘脑增加能量代谢并减少体重。然而，脂联素是否通过作用于下丘脑调控骨代谢，目前国内外尚无文献报道。本研究在野生型和脂联素基因敲除小鼠第三脑室灌注脂联素或APPL1 shRNA慢病毒，应用组织形态学和micro-CT观察股骨远中端骨量改变情况；体外用浓度梯度脂联素处理下丘脑神经元，通过实时定量PCR、免疫印迹、免疫组化等方法，探讨脂联素影响下丘脑神经元的分子学机制。通过体内、体外两方面研究，拟初步探讨脂联素通过中枢神经系统参与骨代谢调控的分子学机制和信号转导通路。

脂联素（adiponectin）是脂肪组织分泌的具有抗糖尿病和促胰岛素增敏、调控食欲和能量代谢等作用的激素。以往的研究表明下丘脑腹内侧核（ventromedial hypothalamic nucleus， VMH）通过激活下游交感神经，上调成骨细胞RANKL表达，诱导破骨细胞分化和增殖，进而促进骨吸收。近来研究发现脂联素可作用于下丘脑增加能量代谢并减少体重。然而，脂联素是否通过作用于下丘脑调控骨代谢，目前国内外尚无文献报道。本研究在野生型和脂联素基因敲除小鼠第三脑室灌注脂联素或APPL1 shRNA慢病毒，应用组织形态学和micro-CT观察股骨远中端骨量改变情况；体外用浓度梯度脂联素处理下丘脑神经元、人脂肪间充质干细胞、人牙槽骨骨髓间充质干细胞、骨髓间充质干细胞或成骨细胞前体细胞，通过实时定量PCR、免疫印迹、免疫组化等方法，探讨脂联素影响下丘脑神经元的分子学机制。通过体内、体外两方面研究，拟初步探讨脂联素通过中枢神经系统参与骨代谢调控的分子学机制和信号转导通路。脂联素（fAPN和gAPN）第三脑室灌注后促进实验小鼠外周骨量的增加，并明确了脂联素通过作用于下丘脑，通过激活下丘脑神经元APPL1的激活，选择性上调下丘脑TPH2、CART、Htr2C基因的表达，下调CB1基因的表达，降低外周交感神经兴奋性，促进骨髓间充质干细胞成骨向分化。体外用浓度梯度脂联素或APPL1 shRNA慢病毒等，处理下丘脑神经元细胞系mHypoE-N的变化与体内结果一直，再次验证了脂连素中枢骨改建作用的分子学机制和信号转导通路。在外周，脂连素通过激活APPL1/PIK3/Akt/MAPK信号通路，或抑制PTEN信号通路，促进人脂肪间充质干细胞、人牙槽骨骨髓间充质干细胞、骨髓间充质干细胞或成骨细胞前体细胞成骨向分化。综上所述，脂连素通过中枢或外周双重调控作用参与骨代谢过程，本研究结果为脂联素应用于临床治疗骨代谢类疾病提供实验依据。