Lipeng Sun,1 Shouxiang Kuang,1 Yang Li,1 Guodong Wang,1 Jianmin Sun,1 Fengge Zhou,2,* Chenggui Zhang1,* 

1Department of Orthopaedics, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, People’s Republic of China; 2Tumor Research and Therapy Center, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Chenggui Zhang, Department of Orthopaedics, Shandong Provincial Hospital Affiliated to Shandong First Medical University, 9677 Jingshi Road, Jinan, Shandong, 250000, People’s Republic of China, Tel +86 531 6877 3207, Email chenggui1214@pku.edu.cn Fengge Zhou, Email zfg1991125@163.com

Background: Rapid bone loss after fracture elevates the risk of subsequent fractures, but the mechanisms remain unclear. IL-6, a key cytokine involved in fracture healing, is markedly upregulated during the immune response after fracture; however, its role in systemic skeletal deterioration remains poorly defined.
Methods: In this study, we employed label-free proteomics to identify candidate mediators in vertebral samples following fracture. Next, osteocyte siRNA knockdown and Stattic (STAT3 phosphorylation inhibitor) inhibition were used to investigate IL-6 related signaling pathways. Subsequently, indirect co-cultures of osteocyte with osteoclast or osteoblast were used to evaluate the effects of the IL-6 pathway on bone resorption and formation. Furthermore, fractured mice were treated with MR16-1 (monoclonal anti-mouse IL-6 receptor antibody) or Stattic. Then, trabecular and cortical bone in vertebrae and femur were evaluated at 4, 14, and 28 days post-fracture, including histological analysis of p-STAT3+ osteocyte, RANKL expression, and bone formation/resorption markers.
Results: : In vitro, IL-6 dose-dependently elevated RANKL and p-STAT3 levels in osteocyte and promoted osteoclast activity in co-culture. These effects were suppressed by Stattic and replicated by STAT3 knockdown. In contrast, co-culture of osteocyte with osteoblast exhibited no significant alterations in osteogenic marker expression upon IL-6 exposure, suggesting negligible effects on osteoblast activity. In vivo, MR16-1 reduced trabecular bone loss in the vertebrae and femur after fracture. It also diminished p-STAT3+ osteocyte, reduced RANKL expression, and suppressed osteoclast activity without impairing osteoblastogenesis. And Stattic produced a comparable reduction in systemic bone loss and osteoclast overactivation.
Conclusion: This study demonstrates that IL-6 drives osteoclast-mediated bone resorption via STAT3-dependent RANKL induction in osteocyte, thereby aggravating post-fracture systemic bone loss. And the findings highlight that modulating the IL-6/STAT3/RANKL axis and targeting osteocyte function may offer a promising therapeutic approach for preventing bone loss and minimizing the risk of fracture recurrence.

Keywords: osteoporosis, fracture healing, fracture risk, osteocyte, RANKL, inflammation