新型RANKL“饵受体"LGR4及增龄改变在镉致骨质疏松中的作用及机制

Cadmium exposure increases the risk of osteoporosis in old people. Our previous studies have shown that cadmium can promote receptor activator for nuclear factor-κ B ligand (RANK) expression in osteoblast and enhance RANKL-induced osteoclast differentiation. However, the exact mechanisms are not clarified. Recent studies showed that leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4) can influence RANKL expression, bind with RANKL as a decoy receptor which consequently inhibit osteoclast differentiation. We speculate that the effects of cadmium on RANKL expression in osteoblast and the promoting role on RANKL-induced osteoclast formation are both related to its effects on LGR4. In the present study, we observed the effects of cadmium on RANKL/LGR4 expression using real-time PCR, western blot and ELISA in osteoblast and osteoclast. The age-related change of RANKL/LGR4 expression in osteoblast and osteoclast and the effects of cadmium on the expression are also investigated by using Replicative senescence model of osteoblast and osteoclast. Subsequently, the expression of signal transducer and activator of transcription 3 (STAT3) and phosphorylated STAT3(p-STAT3) were determined to show the potential mechanism of cadmium on LGR4. The animal model of cadmium exposure was established to confirm the role of cadmium on LGR4/RANKL expression and their age-related change by using immunohistochemical staining(IHC), enzyme cytochemical staining and real-time polymerase chain reaction(RT-PCR). Our study might contribute to reveal the new potential cellular and molecular mechanisms of cadmium-induced osteoporosis.

镉暴露增加老年人骨质疏松风险。我们发现镉促进成骨细胞(OB)核因子κB受体活化因子配基(RANKL)表达及增强RANKL诱导的破骨细胞(OC)分化，但机制不明。研究显示富含亮氨酸重复序列的G蛋白偶联受体4(LGR4)影响OB RANKL表达，并作为饵受体与RANKL结合，抑制其诱导OC分化。我们推测镉促进OB RANKL表达及增强RANKL作用与LGR4有关。本研究建立镉染毒OB/OC模型，采用RT-PCR、WB及ELISA等方法研究RANKL/LGR4表达的变化；建立OB/OC复制性衰老模型，观察RANKL/LGR4表达的增龄改变及镉对其表达的影响；通过STAT3(pSTAT3)信号通路表达变化，探讨镉影响LGR4表达机制；建立镉染毒动物模型，应用IHC、酶化学染色及RT-PCR等技术在体验证LGR4/RANKL表达及增龄改变。本研究有助揭示镉致骨质疏松的细胞分子机制，完善镉骨损害机理。

环境水平镉暴露致骨质疏松机制尚不明确。我们前期研究发现低剂量镉暴露可以促进破骨细胞形成，并发现核因子κB受体活化因子配体(RANKL)可能具有重要作用。富含亮氨酸重复序列的G蛋白偶联受体4(LGR4)是一种表达于破骨细胞（OC）的、可以和RANKL结合的蛋白。目前Lgr4在镉致骨损害中的作用知之甚少。本研究通过建立不同增龄阶段动物和细胞镉染毒模型，探讨镉骨损害表现，并重点从Lgr4的角度探讨机制。通过饮水染毒建立不同增龄阶段（3 m，12m和18m）大鼠镉暴露（0，10mg/L, 50mg/L）模型，观察对骨量及破骨细胞形成影响，并分析骨组织RANKL/Lgr4基因和蛋白表达。采用原代成骨细胞（OB）和MC3T3-E1细胞株，观察低剂量镉染毒对成骨细胞分化、矿化及Lgr4表达影响。获得原代OC及RAW264.7细胞株诱导获得OC，观察镉（0，30nm/L，60nm/L）对OC数量、体外骨吸收及分泌TRACP5b影响；研究镉作用后RAW264.7细胞中NFATC1及Lgr4表达变化。通过连续传代获得OB增龄模型，研究Lgr4表达的增龄改变及镉的影响。构建STAT3基因沉默载体，转染OB和OC细胞，探讨STAT3信号通路的作用。低剂量镉暴露可导致骨密度下降、骨微结构损伤、骨生物力学性能下降。染毒大鼠OC数量显著高于对照组；随着染毒剂量的增加，胫骨Lgr4阳性细胞逐渐减少，而RANKL阳性细胞显著增加。PCR和WB同样显示Lgr4基因和蛋白表达下降。低剂量镉作用能够刺激OC分化；镉预处理后可以在3d就可以显著促进OC形成；镉作用可以抑制OC Lgr4表达；STAT3基因沉默后可以抑制Lgr4表达，镉暴露可以导致其表达进一步下调。OB衰老和OC增龄过程中Lgr4表达都呈现下降趋势，镉暴露可以导致Lgr4表达进一步降低。同时镉暴露组OC能维持更长的生存时间。本研究从全新的角度解释镉暴露引起OB活性下降和OC过度分化进而导致骨质疏松的共同途径。并揭示STAT3信号途径可能是镉抑制OB和OC Lgr4表达的可能机制。本研究发表SCI收录论文9篇。作为副主编参编相关学术著作一部，参编Academic/ELSEVIER出版社英文图书一部。培养研究生六名。