PD0325901 Alleviates Thrombin-Inhibited Osteogenic Differentiation Through an IL-1β-Activated Feedback Loop between MEK-Erk1/2 and NF-κB Signal Pathways: Insights from Bioinformatics and Experimental Verification

Yang-Shuo Ge^1,2Chun-Meng Huang^1,2Jun Shen^3,4Ting-Ting Meng^1,2Min-Jun Zhao^1,2Jian-Li Yin^1,2Xin-Hui Huang^1,2Liao-Lin Chen^1,2Jia-Hui Luo^1,2Yu-Qing Zhai^1,2Jia-Wei Du^2,5Yi-Lin Wang^2,3Xue-Zong Wang⁵Ping-Ping Sun^1,2*Dao-Fang Ding^1,2*

• ¹Department of Rehabilitation Therapy, The Second Rehabilitation Hospital of Shanghai, Shanghai, China
• ²Institute of Rehabilitation Medicine, Shanghai Academy of Traditional Chinese Medicine, Shanghai, China
• ³Department of Orthopedic, Guanghua Hospital of Integrative Chinese and Western Medicine, Shanghai, China
• ⁴Shanghai Academy of Traditional Chinese Medicine, Arthritis Institute of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China
• ⁵Shi’s Center of Orthopedics and Traumatology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China

Objective: To elucidate the molecular mechanisms underlying thrombin-induced suppression of osteoblast differentiation, and to identify the MEK inhibitor PD0325901 (PD03) as a potential therapeutic candidate. Methods: Following treatment of primary rat osteoblasts with thrombin (20 U/mL) and PD03 (0.1 μM) during osteogenic induction, the cells were harvested and subjected to RNA sequencing to identify differentially expressed genes (DEGs). The combination of network pharmacology and RNA sequencing was used to predict the targets of PD03 in thrombin-induced osteoblasts. Alkaline phosphatase (ALP) activity was assessed through staining and quantitative analysis; the expression of osteogenic genes was measured by quantitative PCR (qPCR) and western blot; mineralized nodule formation was evaluated by Alizarin Red S staining; the expression of signaling pathway-related proteins (p-Erk1/2, p-Stat3, p-p65, MMP-9, COX-2) and proliferation-related proteins (PCNA and MCM2) were examined by western blot; nuclear localization of NF-κB was observed by immunofluorescence (IF); intracellular calcium levels were quantified using a calcium assay and probe labeling; osteoblast proliferation was evaluated by EdU staining; IL-1β secretion in cell supernatants was detected by ELISA; the expression of IL-1RA was measured by western blot; the effects of MMP-9 knockdown and COX-2 overexpression on osteogenic differentiation were investigated. Results: Thrombin promoted osteoblast proliferation and inhibited osteogenic differentiation by upregulating inflammatory factors and activating inflammatory signaling pathways, including MEK-Erk1/2 and NF-κB, which in turn reduced ALP activity, calcium ion influx, expression of osteogenic markers (e.g., Col1α1, Runx2, OCN), and mineralized nodule formation. PD03 reverses these effects by suppressing thrombin-induced activation of IL-1β-dependent signaling pathway, in which the downstream gene MMP-9 plays a critical role. Conclusion: PD03 inhibits thrombin-induced activation of the IL-1β-mediated feedback loop between the MEK-Erk1/2 and NF-κB pathways, thereby restoring bone formation and offering a promising therapeutic approach for mitigating bone loss in patients with elevated thrombin levels.