Oral Citrate Supplementation Mitigates Age-Associated Pathological Intervertebral Disc Calcification in LG/J Mice

Despite the high prevalence of age-dependent intervertebral disc calcification, there is a glaring lack of treatment options for this debilitating pathology. Here, we investigate the efficacy of long-term oral K3Citrate supplementation in ameliorating disc calcification in LG/J mice, a model of spontaneous age-associated disc calcification. K3Citrate successfully reduced the incidence of disc calcification in LG/J mice without deleterious effects on vertebral bone structure, plasma chemistry, and locomotion. Notably, a positive effect on grip strength, a marker of frailty, was evident in treated mice. Spectroscopic investigation of the persisting calcified nodules indicated K3Citrate did not alter the mineral composition, and collagen 10 and aggrecan staining revealed that reactivation of an endochondral differentiation program in endplates may drive LG/J disc calcification. Importantly, K3Citrate reduced calcification incidence without altering the abundance of endplate hypertrophic chondrocytes, suggesting mitigation of disc calcification through Ca2+ chelation. This was further supported by the inability of K3Citrate to inhibit hypertrophic differentiation of chondrogenic ATDC5 cells and minimal effects on their metabolic status. Overall, this study sheds light on the pathogenesis of intervertebral disc calcification in LG/J mice and underscores the therapeutic potential of K3Citrate as a systemic intervention strategy for disc calcification. Overall design: To investigate the impact of the presence (or absence) of intervertebral disc calcification on nucleus pulposus (NP) tissues, caudal NP tissue was collected from 23-month-old LG/J mice from two treatment cohorts: untreated controls, which received regular drinking water; or K3Citrate mice which received water continuously supplemented with 80mM K3Citrate from the time mice were 17 months of age (prior to developing disc calcification) until euthanasia. RNA-sequencing was then conducted on RNA isolated from NP tissues from control and K3Citrate cohorts. Each cohort contains four samples, each corresponding to a different mouse.

尽管年龄依赖性椎间盘钙化的患病率居高不下，但针对这一致残性病理状态的治疗方案却严重匮乏。本研究探究了长期口服枸橼酸钾（K3Citrate）补充疗法对LG/J小鼠（LG/J mice）椎间盘钙化的改善效果，该小鼠品系为自发性年龄相关性椎间盘钙化模型。实验结果表明，枸橼酸钾可有效降低LG/J小鼠的椎间盘钙化发生率，且不会对椎体骨结构、血浆生化指标及运动功能产生不良影响。值得关注的是，给药小鼠的抓握力——这一衰弱标志物——得到了显著改善。对残留钙化结节的光谱学分析显示，枸橼酸钾并未改变其矿物组成；而胶原蛋白X与聚集蛋白聚糖染色结果证实，终板软骨内分化程序的重新激活可能是LG/J小鼠椎间盘钙化的驱动因素。尤为关键的是，枸橼酸钾在降低钙化发生率的同时，并未改变终板肥大软骨细胞的丰度，这提示其可能通过钙离子螯合作用缓解椎间盘钙化。这一结论进一步得到了体外实验结果的支持：枸橼酸钾无法抑制软骨源性ATDC5细胞（ATDC5 cells）的肥大分化，且对其代谢状态仅存在极微弱的影响。总体而言，本研究阐明了LG/J小鼠椎间盘钙化的发病机制，并凸显了枸橼酸钾作为椎间盘钙化全身性干预策略的治疗潜力。实验设计：为探究椎间盘钙化的存在与否对髓核（NP）组织的影响，研究人员从两个处理队列的23月龄LG/J小鼠中收集尾部髓核组织：未给药对照组，饮用常规饮用水；枸橼酸钾组，自小鼠17月龄（即出现椎间盘钙化前）起，持续饮用添加80mM枸橼酸钾的饮用水，直至安乐死。随后对两组髓核组织分离得到的RNA进行RNA测序（RNA-sequencing）。每个队列包含4个样本，每个样本对应一只独立的小鼠。