NF-κB/miR-375/CTGF信号介导咖啡因所致子代骨发育异常的血管发生机制

Epidemiological studies showed that osteoporosis exists intrauterine development origins, its mechanism may be associated with poor angiogenesis. Our previous studies confirmed that prenatal caffeine exposure causes fetal endochondral ossification disorder and skeletal growth retardation. Our pre-experimental results showed that prenatal caffeine exposure could lead to increased susceptibility to osteoporosis in IUGR female offspring rats. It was further found that the mRNA expressions of NF-κB and CTGF in the female fetal growth plate were decreased, and poor angiogenesis of H-type vessel in primary ossification center. In summary, we speculate that prenatal caffeine exposure inhibits NF-κB in IUGR fetal cartilage, thereby increasing miR-375 expression, which inhibits CTGF expression and persists until after birth. CTGF suppresses H-type vessel formation by affecting vascular endothelial cell migration, and resulting in endochondral ossification disorder. This project aims to confirm that the poor angiogenesis of H-type vessel mediates intrauterine programming mechanism of endochondral ossification disorder induced by prenatal caffeine exposure in offspring rats, and further confirmed the molecular mechanism of caffeine inhibiting CTGF expression through NF-κB/miR-375. This project has important meaning for guiding prenatal care and exploring early prevention and treatment targets of fetal originated osteoporosis.

流行病学调查表明，骨质疏松症存在宫内发育起源，其发生机制可能与血管发生不良有关。我们前期研究发现，孕期咖啡因暴露可致胎鼠软骨内成骨障碍及骨骼发育迟缓。我们的预实验结果发现，孕期咖啡因暴露可致子代雌性大鼠骨质疏松易感，进一步发现胎鼠生长板软骨NF-κB和CTGF的mRNA表达降低，初级骨化中心骨软骨交界处H型血管形成不良。综上我们推测：宫内咖啡因暴露可通过抑制胎软骨NF-κB，升高miR-375表达，进而抑制CTGF表达并且持续至出生后，通过影响H型血管内皮细胞迁移而使子代大鼠软骨内成骨受抑，最终导致骨质疏松症的发生。本项目拟通过整体动物水平和细胞实验，阐明NF-κB/miR-375/CTGF信号介导咖啡因所致骨发育异常的血管发生机制，为指导孕期健康生活，探寻胎源性骨质疏松症的早期防治靶标有着重要意义。

骨质疏松症是一种常见的骨代谢性疾病，具有骨量减少、骨力学强度减弱和骨折发生率高等特点。我国40岁以上人群骨质疏松症发病率约为25%，且增长趋势明显，将给社会带来巨大的经济负担。流行病学调查表明，骨质疏松症存在宫内发育起源，其发生机制可能与血管发生不良有关。咖啡因广泛存在于日常生活中常见的各种咖啡、茶类、功能饮料及一些镇痛药物中。咖啡因脂溶性较高，可通过胎盘进入胎儿体内，并容易在胎儿体内蓄积。我们前期研究发现，孕期咖啡因暴露（PCE）可致胎鼠软骨内成骨障碍及骨骼发育迟缓。本项目旨在阐明PCE所致子代骨发育异常的血管发生机制。本研究通过动物实验发现，与对照组相比，PCE组胎鼠股骨长度及初级骨化中心长度均较短，矿化面积较小；且PCE组成骨功能相关基因Runx2、Osterix、Osteocalcin、Col 1的mRNA均表达降低。此外，免疫荧光实验显示，PCE组胎鼠长骨初级骨化中心中血管内皮CD31与Emcn同时高表达的细胞数量明显较对照组减少；且生长板内H型血管形成相关基因CTGF mRNA表达降低。同时，通过对子代大鼠出生后miR-375和CTGF、股骨骨量及骨发育相关指标进行检测，初步证实 H 型血管发生不良介导咖啡因所致子代大鼠骨发育异常的发生。进一步，我们在细胞水平发现，咖啡因处理（10μM, 3d）可以导致胎生长板软骨细胞CTGF mRNA及蛋白表达均降低，而miR-375表达升高；将软骨细胞与原代内皮细胞共培养24h后，与对照组相比，咖啡因组内皮细胞成管能力降低。进一步，通过对软骨细胞转染CTGF过表达质粒或者miR375 inhibitor，并进行共培养，我们发现CTGF过表达质粒或者miR375 inhibitor均可以逆转咖啡因所导致的内皮细胞血管形成能力降低。综上提示，PCE通过miR375/CTGF信号所致子代H型血管形成不良，并进一步引起子代骨发育不良。本研究结果阐明了PCE所致H型血管形成减少所致骨发育不良的分子机制，为指导孕期健康，制定胎源性骨质疏松症产前诊断和早期纠正性治疗的靶标，提供了理论和实验依据。