神经肽Y在骨质疏松骨微损伤修复机制中的作用

The accumulation of bone microdamage is a critical risk factor of osteoporotic fracture. Understanding the remodeling mechanisms of osteoporotic bone microdamage may improve the treatment of osteoporosis and the prevention of osteopototic fracture significantly. Our previous studies revealed that the cancellous bones of osteoporotic patients caused by estrogen deficiency increased neuropeptide Y (NPY) expression and the incidence of bone microdamage is higher, which suggested that NPY may participate in the remodeling of osteoporotic bone microdamage through activating its receptors, while the mechanisms remain unclear. In the first part of this study, we will compare the serum and bone samples of different degenerative osteoarthrosis patients, making a preliminary exploration on the relationship of the levels of estrogen and the expression of NPY and receptors; And in the second part, we will apply the rats with osteoblast-specific deletion of the Y1 receptor generated by CRISPR/Cas9 to build the ovariectomized rat model with tibia fatigue loading, further researching the actions of blocking Y1R of osteoblast on bone microdamage, microstructure, biomechanical properties and pain threshold. In the third part of our study, the osteoblast-osteoclast contactless co-culture system will be built. Employing this system, we try to explore whether the Y1R antagonist could indirectly enhance the activity of differentiation and bone absorption of osteoclast through activating cAMP/PKA-RANKL pathway in osteoblast, which facilitates the elimination of bone microdamage. Meanwhile, we investigate if the Y1R antagonist could enhance the osteogenetic activity of osteoblast by blocking Y1R expressed on the surface of osteoblast, which promotes the remodeling of osteoporotic bone microdamage. This study will provide new ideas and theoretical basis in the field of prevention and treatment of osteoporosis.

骨微损伤的积累是骨质疏松性骨折发生的重要因素。研究骨质疏松骨微损伤的修复机制对于治疗骨质疏松及预防骨质疏松性骨折的发生具有重要意义。我们前期研究发现:雌激素缺乏导致骨质疏松患者松质骨内NPY增加,骨微损伤发生率增高,提示我们:NPY通过其受体,参与骨质疏松骨微损伤的修复,但具体机制尚不明确。本课题拟通过比较人体不同的退变性骨关节病,初步明确雌激素水平与NPY及受体表达的关系;采用CRISPR/Cas9技术构建的Y1R基因成骨细胞条件性敲除大鼠进行骨质疏松骨微损伤造模,研究成骨细胞上的Y1R是否对骨微损伤修复、骨微结构、生物力学性能及痛阈产生影响；建立成骨细胞-破骨细胞非接触式共培养体系,揭示Y1R拮抗剂是否通过成骨细胞cAMP/PKA-RANKL途径间接调控破骨细胞分化活性,清除骨微损伤,同时增加成骨细胞活性,促进骨质疏松骨微损伤修复,提高骨强度,为骨质疏松的治疗提出了新的思路和理论依据。

背景:骨质疏松(osteoporosis, OP)是导致骨折危险性增加的全身代谢性骨病。骨微损伤的累积是影响骨质量，引起骨质疏松性骨折的关键因素之一。神经肽Y (neuropeptide Y, NPY)在骨组织中主要与Y1受体(Y1 receptor, Y1R)结合，参与调控骨代谢。目前尚不清楚NPY及Y1R是否参与骨质疏松及骨微损伤的修复。本课题拟从临床研究、动物及细胞实验三个方面探究NPY在骨质疏松骨微损伤修复中的作用及机制。方法:本课题的研究分为三部分。第一部分选择临床上因骨质疏松骨折及骨关节炎(osteoarthritis, OA)行人工髋关节置换术患者各10例。取股骨头样本行骨微结构、生物力学以及骨微损伤检测,通过免疫组化检测两组NPY及Y1R的表达差异。第二部分使用Y1R拮抗剂对骨质疏松骨微损伤模型大鼠进行干预。观察各组大鼠骨微损伤及骨微结构的差异，通过茜素红染色、TRAP染色及Western blot等检测骨形成及骨吸收活性。第三部分采用体外培养原代大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)，在成骨诱导分化的条件下加入外源性NPY、Y1R拮抗剂或PKA激活剂。通过ALP染色、茜素红染色及Western blot等检测BMSCs成骨分化活性以及RANKL/OPG表达情况。结果:第一部分结果显示OP组的骨微结构明显破坏，骨微损伤较OA组明显累积，免疫组化结果显示OP组骨组织中NPY及Y1R的表达均较OA组明显增多；第二部分结果显示Y1R拮抗剂治疗可明显改善去卵巢大鼠的骨质疏松，减少疲劳加载诱发的骨微损伤产生，显著上调骨髓细胞RUNX2和OPG的蛋白表达，并且下调MMP9、Cath-K以及RANKL的蛋白表达。第三部分结果显示在BMSCs的成骨诱导分化过程中，给予外源性的NPY可以显著地抑制BMSCs的成骨分化和RUNX2及OCN的蛋白表达，上调RANKL/OPG，显著抑制cAMP/PKA/CREB通路分子的表达。同时加入Y1R拮抗剂或PKA激动剂培养可部分阻断或逆转NPY的效应。结论: NPY可通过Y1R抑制cAMP/PKA/CREB通路，抑制BMSCs的成骨分化并且上调RANKL/OPG。该效应可能是Y1R拮抗剂调节骨重建平衡，从而促进骨质疏松骨微损伤修复的重要作用机制。