模拟失重条件下免疫调节肽NPFF对破骨细胞分化的影响

The role of osteoclasts in weightlessness-induced bone loss has received increasing attention. Enhanced osteoclast differentiation plays a pivotal role in the process of weightlessness-induced bone loss. Neuropeptide FF (NPFF) is one kind of highly conserved endogenous neuropeptide (octapeptide) with hormone-like activity. Our preliminary findings suggest that NPFF inhibited osteoclast formation, nitric oxide (NO) release and expression of osteoclast-related genes in RANKL-treated RAW264.7 cells. Moreove, NPFF receptor (NPFF receptor 2, NPFFR2) was naturally expressed in RAW264.7 cells. Furthermore, NPFF modulated the viability, NO release and NF-κB activation of RAW264.7 cells via NPFFR2-dependent manner. In addition, simulated weightlessness exposure enhanced osteoclast differentiation and NPFFR2 expression level in RAW264.7 cells. Based on these findings, we speculated that NPFF may regulate osteoclast differentiation under conditions of simulated weightlessness..In the present project, the modulatory effect of NPFF upon osteoclast differentiation will be investigated both in vitro and in vivo. 1) In vitro, the influence of NPFF upon osteoclast differentiation under conditions of simulated weightlessness will be evaluated, which includes osteoclast formation, bone-resorbing ability, osteoclast markers, osteoclast-related gene levels and osteoclast signal pathways (NO and NF-κB). Moreover, the involvement of NPFF receptor in osteoclast differentiation will also be examined. 2) In vivo, the effect of NPFF on hindlimb-suspended mouse will be evaluated, including primary osteoclast formation, bone structure, mechanical property, osteoclast-related gene levels and signal pathways (NO and NF-κB). Taken together, our study will provide useful evidence for further exploitation of mechanisms underlying weightlessness-induced bone loss by using theory of osteoimmunology, which may open new avenue of potential therapeutic targets for weightlessness-caused bone loss..

破骨细胞是骨吸收的主要功能细胞，失重条件下破骨细胞分化能力增强导致骨吸收增加是引起骨质流失的重要原因之一。免疫调节肽NPFF是哺乳动物体内高度保守的具有激素样活性的内源性神经肽（八肽）。申请者发现：NPFF抑制破骨细胞形成、一氧化氮（NO）和骨吸收相关基因的表达；NPFF通过天然表达于前破骨细胞的受体调节细胞活力、NO和NF-κB信号通路；模拟失重促进破骨细胞分化，上调NPFF受体表达。因此提出：模拟失重下NPFF可能调节破骨细胞的分化。.本项目拟从细胞和整体动物水平研究：1）模拟失重条件下NPFF对破骨细胞形成、骨吸收功能及相关基因、NO和NF-κB信号通路的影响，以及NPFF受体在破骨细胞分化过程中的作用；2）NPFF对尾吊小鼠骨组织破骨细胞形成、骨组织结构、力学性能、骨吸收相关基因和信号通路（NO和NF-κB）的影响。为骨免疫学在失重性骨质流失发生中的分子机制提供实验依据和新靶点。

破骨细胞作为骨吸收的主要功能细胞，其发挥生理功能的过程包括破骨细胞分化形成、骨吸收等过程。失重条件下，破骨细胞分化能力增强导致骨吸收增加是引起骨质流失的重要原因之一。但机制尚未明确。免疫调节肽NPFF是哺乳动物体内高度保守的具有激素样活性的内源性神经肽（八肽）。申请者前期研究发现：NPFF抑制通过其受体NPFFR2抑制破骨细胞分化。.本项目以前破骨细胞RAW264.7（体外）和后肢尾悬吊（体内）为实验材料，采用模拟失重条件（RPM和抗磁悬浮），开展了：（1）模拟失重条件下NPFF对破骨细胞形成、骨吸收功能及相关基因、NO和NF-κB信号通路的影响，以及NPFF受体在破骨细胞分化过程中的作用；（2）NPFF对尾吊小鼠骨组织破骨细胞形成、骨组织结构、力学性能、骨吸收相关基因和信号通路（NO和NF-κB）的影响。.研究发现：（1）模拟失重条件下破骨细胞形成增加，破骨标志基因（如TRAP、MMP-9、CathK）表达升高、细胞骨架增大、且骨吸收功能增强，信号分子检测发现NO参与破骨细胞分化的调节，提示NO可能是促进破骨细胞分化的信号分子之一。（2）NPFF抑制模拟失重引起的破骨细胞分化成熟，破骨细胞形成减少，破骨标志性基因表达下降，骨吸收功能下降，且NO信号通路被抑制，表明NO信号通路参与NPFF对破骨细胞分化的调节。（3）在NPFFR2沉默的RAW264.7细胞上，NPFF水平对破骨细胞分化的抑制作用减弱，表明NPFFR2参与该过程。（4）在后肢尾悬吊小鼠上，NPFFR2的表达上升，提示NPFFR2参与该过程。NPFF抑制破骨细胞形成，增加骨密度，提示NPFF可对抗后肢尾悬吊处理引起小鼠承重骨骨质疏松。.本课题研究进展顺利，圆满完成了课题任务书规定的研究内容，发表研究论文6篇（5篇SCI，1篇中文核心），会议论文3篇。滚动申获科研基金6项（含国家自然科学基金1项）。参加国际学术会议3次，参加国内会议10次。培养青年科技骨干1名，博士后2名，博士研究生3名，硕士研究生1名。