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

Thrombin transcriptionally induces inflammatory genes and suppresses osteogenic markers via RNA-seq identified pathways. The MEK inhibitor PD0325901 (PD03) is identified to be a key modulator targeting the IL-1ß-MMP-9 axis. Our finding highlights that PD03 inhibits thrombin-induced suppression of osteoblast differentiation through repressing IL-1ß-mediated MEK-Erk1/2 and NF-?B feedback loop and restoring osteogenic gene expression. Overall design: The experimental design involved isolating primary osteoblasts from the calvaria of 24-hour-old male Sprague-Dawley rats using 0.1% collagenase type I digestion, culturing them in high-glucose DMEM with 10% fetal bovine serum and penicillin-streptomycin, and using first-passage cells for analyses; for osteogenic differentiation, cells at 75% confluence were induced in medium containing 10% fetal bovine serum, 10 mM ß-glycerophosphate, 50 µg/mL L-ascorbic acid, and 10^{-7} M dexamethasone, with groups including control (induction medium alone), thrombin (20 U/mL thrombin), PD03 (20 U/mL thrombin + 0.1 µM PD0325901), C188 (20 U/mL thrombin + 5 µM C188-9), QNZ (20 U/mL thrombin + 0.1 µM QNZ), and Vorapaxar (20 U/mL thrombin + 0.1/0.5/1 µM Vorapaxar), inducing for 7 days to assess gene expression or 21 days for mineralization; transient transfection used Lipofectamine 2000 with pCDH-GFP-COX-2 plasmid or MMP-9 siRNA, verifying efficiency by western blot and evaluating osteogenic markers after 7 days; RNA-seq on 7-day induced cells (three replicates per group) involved library construction on Illumina HiSeq 4000.