淫羊藿总黄酮调控MITF/Rab27A依赖的破骨细胞外泌体分泌促进慢性骨髓炎抗感染后的骨修复机制研究

Chronic osteomyelitis is mainly caused by traumatic infection. Antibiotic is used for anti-infection treatment, however, bone repair is difficult to treat after anti-infection treatment. Exosomes regulate intercellular communication between osteoclasts and osteoblasts, which is related to bone repair. MITF/Rab27A is an important signaling pathway in regulating exosomes secretion from osteoclasts. The exosomes derived from ostoeclasts transduct miRNA-214-3p into osteoblasts, and the target genes of miRNA-214-3p is ATF4. ATF4 modulate the expression of osteocalcin along with Runx2. In the previous study, total flavonoids of Herba Epimedii (TFE) were proved to promote bone repair after anti-infection treatment in the chronic osteomyelitis model rabbits. On the other hand, TFE inhibited the mRNA expression of MITF in osteoclasts, and the quantities of exosomes derived from osteoclasts. TFE also reduced expression of miRNA-214-3p in exosomes derived from osteoclasts. This research aim to explore the hypothesis "the total flavonoids of Herba Epimedii modulate the exosomes secretion from osteoclasts via MITF/Rab27A-dependent pathway, and then the exosomes modulate miR-214-3p/ATF4/Runx2 signaling axis, which induce osteocalcin expression". We will use exosomes identification and imaging, gene over expression, and gene silencing technologies to verified this hypothesis. This study aim to explain the mechnism of bone repair after anti-infection treatment of chronic osteomyelitis by using TFE based on exosomes modulation. This study will provide a new direction of bone repair by using of bone strenthen Traditional Chinese Medicine.

慢性骨髓炎多由创伤感染所致，采用抗生素抗感染治疗后骨修复困难，尚无解决方法。外泌体可调控破骨-成骨的细胞间通讯进而影响骨修复。MITF/Rab27A是破骨细胞中调控外泌体分泌到胞外的重要途径，且破骨细胞外泌体可转导miRNA-214-3p进入成骨细胞，调控靶基因ATF4，ATF4与Runx2协同调控骨钙素的表达。申请者研究发现，淫羊藿总黄酮可促进慢性骨髓炎兔抗感染治疗后的骨修复；抑制破骨细胞MITF的表达水平以及外泌体的数量，并抑制外泌体miRNA-214-3p表达水平。本课题运用外泌体鉴别和成像、基因过表达沉默等技术，探究“淫羊藿总黄酮调控MITF/Rab27A依赖的破骨细胞外泌体分泌，进而调控miR-214-3p/ATF4/Runx2信号轴，促进成骨细胞骨钙素表达”的科学假说。阐明基于外泌体介导的淫羊藿总黄酮促进慢性骨髓炎抗感染治疗后骨修复的科学本质，为健骨中药促进骨修复提供新的思路。

慢性骨髓炎多由创伤感染所致，采用抗生素抗感染治疗后骨修复困难，尚无解决方法。外泌体可调控破骨-成骨的细胞间通讯进而影响骨修复。MITF/Rab27A是破骨细胞中调控外泌体分泌到胞外的重要途径，且破骨细胞外泌体可转导miRNA-214-3p进入成骨细胞，调控靶基因ATF4，ATF4与Runx2协同调控骨钙素的表达。本项目通过UPLC-TOF/MS方法分析淫羊藿总黄酮主要成分及血中移行成分，明确淫羊藿总黄酮主要药效成分为淫羊藿苷与宝藿苷；采用改良的慢性骨髓炎模型制备方法制备慢性骨髓炎兔以及大鼠模型，抗感染治疗后灌胃给予淫羊藿总黄酮，采用骨组织病理，免疫组化等手段，发现淫羊藿总黄酮改善局部骨组织骨密度及骨小梁厚度、数量，且调控局部骨组织周围破骨细胞及成骨细胞的功能，对破骨细胞外泌体分泌关键机制MITF/Rab27A有调控作用。通过RANKL诱导骨髓间充质干细胞培养破骨细胞，采用差速离心等方法制备破骨细胞外泌体，发现淫羊藿总黄酮通过MITF/Rab27A机制调控破骨细胞外泌体的分泌。将破骨细胞外泌体与成骨细胞共培养，发现淫羊藿总黄酮调控破骨细胞外泌体，进而调控成骨细胞miRNA-214-3p/ATF4/Runx2信号轴，影响成骨细胞增殖和矿化的机制。本项目的开展明确淫羊藿总黄酮促进慢性骨髓炎抗感染治疗后的骨修复作用并发现其作用靶点，为慢性骨髓炎抗感染治疗后的骨愈合提供新的治疗方法。